Alkoxy substituted imidazo[1,2-a]pyridines having affinity for amyloid

ABSTRACT

The present invention provides a compound which has affinity with amyloid, shows sufficiently rapid clearance from normal tissues and is suppressed in toxicity such as mutagencity. Provided is a compound represented by the following formula (1) or a salt thereof: 
                         
wherein A 1 , A 2 , A 3  and A 4  independently represents a carbon or nitrogen; R 1  is a halogen substituent; R 2  is a halogen substituent; and m is an integer of 0 to 2, provided that at least one of R 1  and R 2  is a radioactive halogen substituent, at least one of A 1 , A 2 , A 3  and A 4  represents a carbon, and R 1  binds to a carbon represented by A 1 , A 2 , A 3  or A 4  as well as a low-toxic diagnostic agent comprising a compound represented by the preceding formula or a salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a U.S. National Phase of International ApplicationPCT/JP2007/059922, filed May 15, 2007, and claims the benefit of foreignpriority under 35 U.S.C. §119 based on JP 2006-140044, filed May 19,2006, and JP 2006-324701, filed Nov. 30, 2006, the entire disclosures ofwhich applications are hereby incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a compound for use in diagnosis ofcerebral degenerative disease. More specifically, the invention relatesto a compound useful for amyloid detection at lesion sites in diagnosisof Alzheimer's disease and other diseases with amyloid accumulation.

BACKGROUND ART

Diseases with the onset of deposition of a fibrous protein calledamyloid in various organs or tissues in bodies are generally referred toas amyloidosis. A feature common to amyloidosis is that the fibrousprotein called amyloid which is enriched with the β-sheet structure isdeposited at various organs systemically or at sites topically so thatfunctional abnormalities are triggered in the organs or tissues.

Alzheimer's disease (hereinafter referred to as AD), which is a typicalamyloidosis disease, is known as a disease causing dementia. Thisdisease is lethal with progressive deposition of amyloid in brain, andthus is said to be a disease that causes concern in society comparedwith other amyloidosis diseases. In recent years, the number of ADpatients is rapidly increasing in developed countries with agingsocieties, thereby causing a social problem.

From the pathohistological viewpoint, AD is characterized by threepathological findings in brain, namely development of senile plaques,formation of neurofibrillary tangles, and extensive neuronal loss. Thesenile plaque has a structure mainly composed of amyloid, and is said toappear at the earliest stage of AD onset and thus is pathologicallyfound in brain about 10 or more years before appearance of clinicalsymptoms.

AD is diagnosed by carrying out various evaluations of cognitivefunctions (for example, Hasegawa scale, ADAS-JCog and MMSE) in auxiliarycombination with imaging diagnosis such as CT and MRI. However, themethod based on such evaluations of cognitive functions is low indiagnostic sensitivity at the early stage of the onset, and isfurthermore problematic in that diagnostic results are susceptible toinborn cognitive functions of individuals. At present, it is practicallyimpossible to establish a definite diagnosis of AD while an AD patientis still alive, because the definite diagnosis requires a biopsy of alesion (Non-Patent Document 1).

Meanwhile, a report tells that amyloid constituting senile plaques is anaggregate of amyloid β protein (hereinafter referred to as Aβ). Also,numerous reports tell that the Aβ aggregate forms a β-sheet structurethat causes nerve cell toxicity. Based on these findings, the so-called“Amyloid Cascade Hypothesis” is proposed, which suggests that cerebraldeposition of Aβ triggers the downstream phenomena, namely, formation ofneurofibrillary tangles and neuronal loss (Non-Patent Document 2).

Based on these facts, attempts have recently been made to detect AD invivo using a compound having high affinity with amyloid as a marker.

Many of such probes for imaging diagnoses of cerebral amyloid arehydrophobic low-molecular compounds that are high in affinity withamyloid and high in cerebral transferability and are labeled withvarious radioactive species such as ¹¹C, ¹⁸F and ¹²³I. For example,reports tell ¹¹C or radioactive halogen labeled forms of compoundsincluding various thioflavin derivatives such as6-iodo-2-[4′-(N,N-dimethylamino)phenyl]benzothiazole (hereinafterreferred to as TZDM) and6-hydroxy-2-[4′-(N-methylamino)phenyl]benzothiazole (hereinafterreferred to as 6-OH-BTA-1) (Patent Document 1, Non-Patent Document 3);stilbene compounds such as (E)-4-methylamino-4′-hydroxystilbene(hereinafter referred to as SB-13) and(E)-4-dimethylamino-4′-iodostilbene (hereinafter referred to as m-I-SB)(Patent Document 2, Non-Patent Document 4, Non-Patent Document 5);benzoxazole derivatives such as6-iodo-2-[4′-(N,N-dimethylamino)phenyl]benzoxazole (hereinafter referredto as IBOX) and6-[2-(fluoro)ethoxy]-2-[2-(2-dimethylaminothiazol-5-yl)ethenyl]benzoxazole(Non-Patent Document 6, Non-Patent Document 7), DDNP derivatives such as2-(1-{6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile(hereinafter referred to as FDDNP) (Patent Document 4, Non-PatentDocument 8); and imidazopyridine derivatives such as6-iodo-2-[4′-(N,N-dimethylamino)phenyl]imidazo[1,2-a]pyridine(hereinafter referred to as IMPY) (Patent Document 3, Non-PatentDocument 9). Further, some of these probes for imaging diagnosis havebeen studied on human imaging and have been reported to show asignificant accumulation in AD patient's brain compared with normalpersons (Non-Patent Document 10, Non-Patent Document 11, Non-PatentDocument 12, Non-Patent Document 13).

-   [Patent Document 1] JP-T-2004-506723-   [Patent Document 2] JP-T-2005-504055-   [Patent Document 3] JP-T-2005-512945-   [Patent Document 4] JP-T-2002-523383-   [Non-Patent Document 1] J. A. Hardy & G. A. Higgins, “Alzheimer's    Disease: The Amyloid Cascade Hypothesis.”, Science, 1992, 256, p.    184-185-   [Non-Patent Document 2] G. McKhann et al., “Clinical diagnosis of    Alzheimer's disease: Report of the NINCDS-ADRDA Work Group under the    auspices of Department of Health and Human Services Task Force on    Alzheimer's Disease.”, Neurology, 1984, 34, p. 939-944-   [Non-Patent Document 3] Z.-P. Zhuang et al., “Radioiodinated    Styrylbenzenes and Thioflavins as Probes for Amyloid    Aggregates.”, J. Med. Chem., 2001, 44, p. 1905-1914-   [Non-Patent Document 4] Masahiro Ono et al., “11C-labeled stilbene    derivatives as Aβ-aggregate-specific PET imaging agents for    Alzheimer's disease.”, Nuclear Medicine and Biology, 2003, 30, p.    565-571-   [Non-Patent Document 5] H. F. Kung et al., “Novel Stilbenes as    Probes for amyloid plaques.”, J. American Chemical Society, 2001,    123, p. 12740-12741-   [Non-Patent Document 6] Zhi-Ping Zhuang et al.,    “IBOX(2-(4′-dimethylaminophenyl)-6-iodobensoxazole): a ligand for    imaging amyloid plaques in the brain.”, Nuclear Medicine and    Biology, 2001, 28, p. 887-894-   [Non-Patent Document 7] Furumoto Y et al., “[¹¹C]BF-227: A New    ¹¹C-Labeled 2-Ethenylbenzoxazole Derivative for Amyloid-β Plaques    Imaging.”, European Journal of Nuclear Medicine and Molecular    Imaging, 2005, 32, Sup. 1, P759-   [Non-Patent Document 8] Eric D. Agdeppa et al.,    “2-Dialkylamino-6-Acylmalononitrile Substituted Naphthalenes (DDNP    Analogs): Novel Diagnostic and Therapeutic Tools in Alzheimer's    Disease.”, Molecular Imaging and Biology, 2003, 5, p. 404-417-   [Non-Patent Document 9] Zhi-Ping Zhuang et al., “Structure-Activity    Relationship of Imidazo[1,2-a]pyridines as Ligands for Detecting    β-Amyloid Plaques in the Brain.”, J. Med. Chem., 2003, 46, p.    237-243-   [Non-Patent Document 10] W. E. Klunk et al., “Imaging brain amyloid    in Alzheumer's disease with Pittsburgh Compound-B.”, Ann. Neurol.,    2004, 55, p. 306-319-   [Non-Patent Document 11] Nicolaas P. L. G. Verhoeff et al., “In-Vivo    Imaging of Alzheimer Disease β-Amyloid With [11C]SB-13 PET.”,    American Journal of Geriatric Psychiatry, 2004, 12, p. 584-595-   [Non-Patent Document 12] Hiroyuki Arai et al., “[11C]-BF-227 AND PET    to Visualize Amyloid in Alzheimer's Disease Patients”, Alzheimer's &    Dementia: The Journal of the Alzheimer's Association, 2006, 2, Sup.    1, S312-   [Non-Patent Document 13] Christopher M. Clark et al., “Imaging    Amyloid with 1123 IMPY SPECT”, Alzheimer's & Dementia: The Journal    of the Alzheimer's Association, 2006, 2, Sup. 1, S342

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

As described above, various compounds are disclosed as probes forimaging diagnosis for amyloid, and researched for clinical application.

Experiments in normal mice show that [125]-labeled TZDM, IBOX and m-I-SBare all transferred into brain 2 minutes after administration. However,these compounds are insufficient in clearance from normal tissues, andtend to accumulate gradually in brain as time passes afteradministration (JP-T-2005-512945; Zhi-Ping Zhuang et al., NuclearMedicine and Biology, 2001, 28, p. 887-894; H. F. Kung et al., J. Am.Chem. Soc., 2001, 123, p. 12740-12741). When the clearance from normaltissues is insufficient, a problem arises in that sufficient contrastcannot be obtained at amyloid accumulation sites. [¹¹C]-labeled SB-13shows a clearance from normal tissues in experiments in rats, however,it cannot be said that the clearance is sufficiently fast (Masahiro Onoet al., Nuclear Medicine and Biology, 2003, 30, p. 565-571).

Meanwhile, it is revealed that compounds having an imidazopyridineskeleton such as IMPY have a property of transferring to brain andaccumulating at amyloid after administration, and also have an excellentproperty of rapid clearance from normal tissues unlike theabove-described compounds, as a result of experiments using[¹²⁵I]-labeled compounds. However, IMPY is a compound positive inreverse mutation test. In order to use this compound as a probe forimaging diagnosis, sufficient care must be taken about dosage andadministration manner. (International Publication WO03/106439 pamphlet)

FDDNP is also reported to be positive in reverse mutation test.(International Publication WO03/106439 pamphlet)

A preferable probe targeting amyloid for imaging diagnosis would be acompound that is excellent in affinity with amyloid and sufficientlyrapid in clearance from normal tissues like IMPY but is suppressed intoxicity such as mutagenicity. However, no compound with such propertieshas been disclosed.

Furthermore, in accordance with results of our studies (refers toComparative Example II-6 described later), it has been confirmed thatIMPY accumulates unspecifically on white matter or other sites whereamyloid is not deposited. As an AD diagnostic agent, a compound must beused which is suppressed in unspecific accumulation on sites other thanamyloid deposition, but such a compound has not been disclosed.

The present invention has been made under such circumstances wherevarious compounds as probes targeting amyloid for imaging diagnosis havebeen disclosed but there has been no compound which is confirmed to havea clinically tolerable property, and aims at providing a compound thathas affinity with amyloid, exhibits sufficiently rapid clearance fromnormal tissues and further is suppressed in toxicity such asmutagenicity.

Means for Solving the Problems

The inventors have found that a group of compounds satisfying theabove-described requirements can be obtained from a compound with animidazopyridine-phenyl skeleton or a skeleton similar thereto whosephenyl group has a carbon atom to which an oxygen atom is attached, andthus have completed the present invention.

Specifically, according to one aspect of the present invention, acompound represented by the following formula (1):

or a salt thereof, and a low-toxic diagnostic agent for Alzheimer'sdisease comprising a compound represented by the above formula (1) or asalt thereof are provided.

In the formula (1), R¹ and R² are halogen substituents, and at leasteither of them is a radioactive halogen substituent. Various elementscan be used as the halogen, and fluorine, bromine or iodine can bepreferably used. As the radioactive halogen, can be used variouselements, preferably a halogen selected from ¹⁸F, ⁷⁶Br, ¹²³I, ¹²⁴I, ¹²⁵Ior ¹³¹I, and more preferably a halogen selected from ¹⁸F, ¹²³I or ¹²⁵I.

A₁, A₂, A₃ and A₄ independently represent a carbon or nitrogen, and itis necessary that at least one of these represents a carbon. Preferably,3 or more of A₁, A₂, A₃ and A₄ represent carbons, and more preferably,all of them represent carbons. In the formula (1), R¹ binds to a carbonrepresented by A₁, A₂, A₃ or A₄.

In addition, m is an integer of 0 to 2. A binding site for R¹ ispreferably a carbon represented by A₃, that is, a carbon at 6-position.

According to another aspect of the present invention, a compoundrepresented by the following formula (2):

or a salt thereof is provided.

In the formula (2), R³ is a group selected from the group consisting ofa non-radioactive halogen substituent, nitro substituent,trialkylstannyl substituent having alkyl chains with 1 to 4 carbon atomsand triphenylstannyl substituent. As the trialkylstannyl substituent,various substituents can be used, and trimethylstannyl substituent andtributylstannyl substituent are preferably used.

R⁴ is a group selected from the group consisting of a non-radioactivehalogen substituent, methanesulfonyloxy substituent,trifluoromethanesulfonyloxy substituent and aromatic sulfonyloxysubstituent. As an aromatic sulfonyloxy substituent, toluenesulfonicacid, nitrobenzenesulfonic acid and benzenesulfonic acid can bepreferably used.

As a non-radioactive halogen substituent of R³ and R⁴, various halogenscan be used, but preferably a halogen capable of being a target ofnucleophilic substitution reactions using a radioactive fluorine or ahalogen capable of being a target of isotope exchange reactions with aradioactive iodine can be used, and more preferably chlorine, iodine orbromine can be used. At least one of R³ and R⁴ is preferably thenon-radioactive halogen substituent.

A₅, A₆, A₇ and A₈ independently represent a carbon or nitrogen, and itis necessary that at least one of these represents a carbon. Preferably,3 or more of A₅, A₆, A₇ and A₈ represent carbons, and more preferably,all of them represent carbons. In the formula (2), R³ binds to a carbonrepresented by A₅, A₆, A₇ or A₈

In addition, n is an integer of 0 to 2.

Further, according to the present invention, there is provided acompound with an imidazopyridine-phenyl skeleton in which a carbon atomat 4′-position of the phenyl group is bonded via an oxygen atom to analkyl chain which is substituted or non-substituted at the terminalthereof. A binding site for R³ is preferably a carbon represented by A₇,that is, a carbon at 6-position.

Specifically, according to still another aspect of the presentinvention, there are provided a compound represented by the followingformula (3):

or a salt thereof, and a low-toxic diagnostic agent for Alzheimer'sdisease comprising a compound represented by the above formula (3) or asalt thereof. Specifically, a compound represented by the above formula(3) or a salt thereof provides a low-toxic and highly-specificdiagnostic agent for Alzheimer's disease. A low-toxic andhighly-specific diagnostic agent for Alzheimer's disease here refers toa diagnostic agent which has a property of accumulating at amyloid andhardly accumulating at other sites or rapidly clearing other sites evenif it accumulates there, and thus shows high specificity of amyloidimaging in a certain period of time after administration.

In the formula (3), R⁵ is a radioactive halogen substituent. As R⁵, canbe used various radioactive halogens, preferably a radioactive halogenselected from the group consisting of ¹⁸F, ⁷⁶Br ¹²³I, ¹²⁴I, ¹²⁵I and¹³¹I, and more preferably ¹⁸F or ¹²³I.

R⁶ is a group selected from the group consisting of hydrogen, hydroxylgroup, methoxy group, carboxyl group, amino group, N-methylamino group,N,N-dimethylamino group and cyano group. R⁶ is preferably hydrogen,hydroxyl group, carboxyl group or amino group, more preferably hydrogenor hydroxyl group, and particularly preferably hydroxyl group.

A₉, A₁₀, A₁₁ and A₁₂ independently represent a carbon or nitrogen, andit is necessary that at least one of these represents a carbon.Preferably, 3 or more of A₉, A₁₀, A₁₁ and A₁₂ represent carbons, andmore preferably, all of them represent carbons. In the formula (3), R⁵binds to a carbon represented by A₉, A₁₀, A₁₁ or A₁₂. In addition, abinding site for R⁵ is preferably a carbon represented by A₁₁, that is,a carbon at 6-position.

Further, p is an integer of 0 to 2.

According to further still another aspect of the present invention, acompound represented by the following formula (4):

or a salt thereof is provided.

In the formula (4), R⁷ is a group selected from the group consisting ofa non-radioactive halogen substituent, nitro substituent,trialkylammonium group having alkyl chains with 1 to 4 carbon atoms,trialkylstannyl substituent having alkyl chains with 1 to 4 carbon atomsand triphenylstannyl group. As a non-radioactive halogen substituent, ahalogen capable of being a target of nucleophilic substitution reactionsusing a radioactive fluorine or a halogen capable of being a target ofisotope exchange reactions with a radioactive iodine can be used, andpreferably chlorine, iodine or bromine can be used. As a trialkylstannylsubstituent, various substituents can be used, and trimethylstannylsubstituent and tributylstannyl substituent are preferably used.

R⁸ is a group selected from the group consisting of hydrogen, hydroxylgroup, methoxy group, carboxyl group, amino group, N-methylamino group,N,N-dimethylamino group and cyano group. R⁸ is preferably hydrogen,hydroxyl group, carboxyl group or amino group, more preferably hydrogenor hydroxyl group, and particularly preferably hydroxyl group.

A₁₃, A₁₄, A₁₅ and A₁₆ independently represent a carbon or nitrogen, andit is necessary that at least one of these represents a carbon.Preferably, 3 or more of A₁₃, A₁₄, A₁₅ and A₁₆ represent carbons, andmore preferably, all of them represent carbons. In the formula (4), R⁷binds to a carbon represented by A₁₃, A₁₄, A₁₅ or A₁₆. In addition, thebinding site for R⁷ is preferably a carbon represented by A₁₅, that is,a carbon at 6-position.

Further, q is an integer of 0 to 2.

Effects of the Invention

The present invention provides a compound that has affinity with amyloidand is sufficiently fast in clearance from normal tissues and suppressedin toxicity such as mutagenicity as well as a diagnostic agent forAlzheimer's disease with low toxicity, and further provides a compoundthat has high affinity with amyloid and is excellent in amyloid imagingin a living body as well as a diagnostic agent for Alzheimer's Diseasehigh in specificity.

BEST MODE FOR CARRYING OUT THE INVENTION I. A Method for Synthesis of aCompound of the Above Formula (1) or (2)

(A Method for Synthesis of a Precursor Compound for a RadioactiveHalogen-labeled Compound)

Hereinafter, a method for synthesis of a precursor compound for aradioactive halogen-labeled compound according to an embodiment of thepresent invention will be described, taking the case of6-tributylstannyl-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine.

First, 4′-hydroxyacetophenone is allowed to react with cupric bromide toprepare 2-bromo-4′-hydroxyacetophenone (FIG. 1-1, Step 1). In thisinstance, the reaction can be conducted in accordance with ordinarymethods, for example, the method described in a literature, King, L.Carroll and Ostrum, G. Kenneth, Journal of organic Chemistry, 1964,29(12), p. 3459-3461.

Then, 2-bromo-4′-hydroxyacetophenone as prepared above is allowed toreact with 2-amino-5-bromopyridine to prepare6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine (FIG. 1-1, Step 2).This step can be done according to the following procedure.

First, 2-bromo-4′-hydroxyacetophenone and 2-amino-5-bromopyridine aredissolved in an inactive solvent such as acetonitrile, and are allowedto react with each other at a reflux temperature for 2 to 6 hours toproduce 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine hydrobromidesalt as white precipitates. The solvent used in this instance may beacetonitrile or another solvent that is usually employed in a similarreaction, for example, methanol and acetone. The reaction temperaturemay be a temperature allowing refluxing, for example, 90° C. when thesolvent is acetonitrile. The amount of the solvent to be used may be anamount sufficient to effect the reaction, however, it should be notedthat if the solvent is too much, it will become difficult to obtainprecipitates of reaction products. For example, when2-bromo-4′-hydroxyacetophenone in an amount corresponding to 10 mmol isused for the reaction, the amount of a solvent to be used can be about40 to 50 mL.

Next, the reaction solution is filtered to recover the precipitates. Thewhite precipitates are suspended in a mixed solution of methanol/water(1:1). Then, an aqueous saturated solutions of sodium hydrogencarbonateis added thereto in a very excessive amount relative to the suspendedprecipitates to release6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine as precipitates. Thenewly generated precipitates are filtered to recover6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine as the targetcompound in this step (FIG. 1-1, Step 2). The amount of the mixedsolution of water/methanol is not specifically limited as long as it issufficient to effect the reaction. However, it should be noted that ifthe amount of the mixed solution is too much, precipitation of productswill be hindered. For example, when 2-bromo-4′-hydroxyacetophenone in anamount corresponding to 10 mmol is used, the mixed solution ofwater/methanol may be used in an amount of about 40 to 100 mL. Theamount of sodium hydrogencarbonate is not specifically limited as longas it is very excessive relative to the above-described precipitates asreaction substrates. For example, when the reaction is effected underthe above-described conditions, the amount of an aqueous saturatedsolution of sodium hydrogencarbonate to be added to the reactionsolution can be about 25 mL.

Then, the 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine preparedabove is sufficiently dried, dissolved in N,N-dimethylformamide, andpotassium carbonate and 3-bromo-1-fluoropropane were added thereto. Thereaction solution is stirred at room temperature for overnight to obtain6-bromo-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine (FIG. 1-1,Step 3). The amount of potassium carbonate to be used may be an amountthat can neutralize hydrobromic acid generated from3-bromo-1-fluoropropane during the reaction, and is typically aboutdouble the other reactant 3-bromo-1-fluoropropane in molar ratio. The3-bromo-1-fluoropropane can be used in an excessive amount relative tothe reaction substrate, and is typically about 1.5 times the reactionsubstrate 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine in molarratio.

The obtained6-bromo-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine wasdissolved in dioxane, and after triethylamine is added, bis(tributyltin)and a catalytic amount of tetrakis-triphenylphosphine palladium areadded. This reaction mixture is heated at about 90° C. and reacted forabout 24 hours, and then a solvent is distilled off and achromatographic purification is performed to obtain6-tributylstannyl-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridineas the target compound (FIG. 1-1, Step 4). The amount ofbis(tributyltin) to be used in this instance may be an amount satisfyinga condition where it is excessive relative to the reaction substrate,specifically, it is about 1.5 times in molar ratio relative to thereaction substrate6-bromo-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine.

When a compound with a substituent at the 6-position being atrialkylstannyl substituent other than tributylstannyl substituent isobtained, various bis(trialkyltin)s that fit purposes can be usedinstead of bis(tributyltin) in Step 4. For example, when a compoundhaving a trimethylstannyl substituent as a substituent at the 6-positionis synthesized, a reaction similar to the above can be performed in Step4 using bis(trimethyltin).

A compound with an imidazopyridine ring in which the binding site forthe functional group is a carbon atom other than the carbon at6-position can be obtained by using a compound with a pyridine ring towhich bromine is bonded at a different site instead of2-amino-5-bromopyridine used in Step 2. For example, when a binding sitefor the functional group is the carbon at 8-position in theimidazopyridine ring, 2-amino-3-bromopyridine may be used instead of2-amino-5-bromopyridine in Step 2.

Further, a precursor compound with a radioactive halogen labeled sitebeing an alkoxyphenyl substituent attached to a carbon atom at2-position of the imidazo pyridine ring can be obtained by using3-bromo-1-propanol instead of 3-bromo-1-fluoropropane, and reacting aresulting compound with p-toluenesulfonylchloride or the like. Forexample,6-bromo-2-[4′-(3″-p-toluenesulfonyloxypropoxy)phenyl]imidazo[1,2-a]pyridinecan be synthesized by the following procedure.

First, 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine prepared aboveis dissolved in N,N-dimethylformamide, and the solution is supplementedwith potassium carbonate and 3-bromo-1-propanol and stirred at roomtemperature for overnight to obtain6-bromo-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine. This isdissolved in pyridine, supplemented with p-toluenesulfonylchloride underan ice bath and then reacted at room temperature to obtain6-bromo-2-[4′-(3″-p-toluenesulfonyloxypropoxy)phenyl]imidazo[1,2-a]pyridineas a target compound. The amount of the p-toluenesulfonylchloride to beused in this instance may be excessive relative to the reactionsubstrate, and is typically about double the reaction substrate6-bromo-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine in molarratio.

A compound in which the alkoxy substituent bound to the phenyl groupthat is bound to the 2-position of the imidazopyridine ring is bound toanother position than the 4′-position, for example,6-tributylstannyl-2-[3′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridinewhich has a fluoropropoxy group at 3′-position, can be synthesized inaccordance with the same reaction as above except using3′-hydroxyacetophenone as a reactant instead of 4′-hydroxyacetophenonein Step 1.

(A Method for Synthesis of a Radioactive Halogen-labeled Compound)

Next, a method for production of a radioactive halogen-labeled compoundaccording to another aspect of the present invention will be described,taking the case of2-[4′-(3″-fluoropropoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine.

For the production of2-[4′-(3″-fluoropropoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine, a[¹²³I]sodium iodide solution to be served for labeling is firstobtained. A [¹²³I]radioactive iodine can be obtained by, for example, aknown method in which a xenon gas is used as a target and exposed toproton bombardment. This [¹²³I]radioactive iodine is made into[¹²³I]sodium iodide solution by using known methods, and used for thelabeling.

Then, the labeling precursor6-tributylstannyl-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridineis dissolved in an inert organic solvent, and a solution of the[¹²³I]iodo bulk dissolved in water, an acid and an oxidizing agent areadded thereto and allowed to react to obtain2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine as a targetcompound. As the inert organic solvent dissolving the precursorcompound, various solvents having no reactivity with the labelingprecursor and [¹²³I]iodo bulk can be used, and preferably methanol canbe used.

As the acid, may be used various ones, and preferably hydrochloric acid.

The oxidizing agent is not particularly limited as long as it can effectthe oxidation of iodine in the reaction solution, and is preferablyhydrogen peroxide or peracetic acid. The amount of the oxidizing agentto be added may be an amount sufficient to oxidize iodine in thereaction solution.

A compound labeled with a radioactive halogen other than iodine can besynthesized by labeling a labeling precursor that fits a purpose ofsynthesis with a radioactive halogen that fits the purpose. For example,in order to synthesize2-[4′-(3″-[¹⁸F]fluoropropoxy)phenyl]-6-bromoimidazo[1,2-a]pyridine, thelabeling precursor6-bromo-2-[4′-(3″-p-toluenesulfonyloxypropoxy)phenyl]imidazo[1,2-a]pyridinecan be reacted with [¹⁸F]fluoride ion in the presence of a phasetransfer catalyst and potassium carbonate.

II. A Method for Synthesis of a Compound of the Above Formula (3) or (4)

(A Method for Synthesis of a Precursor Compound for a RadioactiveHalogen-labeled Compound)

Hereinafter, a method for synthesis of a precursor compound for aradioactive halogen-labeled compound according to an embodiment of thepresent invention will be described, taking the case of6-tributylstannyl-2-[4′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine.

For the production of6-tributylstannyl-2-[4′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine,first, 4′-hydroxyacetophenone is allowed to react with cupric bromide toprepare 2-bromo-4′-hydroxyacetophenone (FIG. 2-1, Step 1). In thisinstance, the reaction can be conducted in accordance with ordinarymethods, for example, the method described in a literature, King, L.Carroll and Ostrum, G. Kenneth, Journal of organic Chemistry, 1964,29(12), p. 3459-3461.

Then, 2-bromo-4′-hydroxyacetophenone as prepared above is allowed toreact with 2-amino-5-iodopyridine to prepare2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine (FIG. 2-1, Step 2).This step can be done according to the following procedure.

First, 2-bromo-4′-hydroxyacetophenone and 2-amino-5-iodopyridine aredissolved in an inactive solvent such as acetonitrile, and are allowedto react with each other at a reflux temperature for 2 to 6 hours toproduce 2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine hydrobromidesalt as white precipitates. The solvent used in this instance may beacetonitrile or another solvent that is usually employed in a similarreaction, for example, methanol and acetone. The reaction temperaturemay be a temperature allowing refluxing, for example, 110° C. when thesolvent is acetonitrile. The amount of the solvent to be used may be anamount sufficient to effect the reaction, however, it should be notedthat if the solvent is too much, it will become difficult to obtainprecipitates of reaction products. For example, when2-bromo-4′-hydroxyacetophenone in an amount corresponding to 10 mmol isused for the reaction, the amount of a solvent to be used can be about40 to 80 mL.

Next, the reaction solution is filtered to recover the precipitates. Thewhite precipitates are suspended in a mixed solution of methanol/water(1:1). Then, an aqueous saturated solution of sodium hydrogencarbonateis added thereto in a very excessive amount relative to the suspendedprecipitates to release2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine as precipitates. Thenewly generated precipitates are filtered to recover2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine as the target compoundin this step (FIG. 2-1, Step 2). The amount of the mixed solution ofmethanol/water is not specifically limited as long as it is sufficientto effect the reaction. However, it should be noted that if the amountof the mixed solution is too much, precipitation of products will behindered. For example, when 2-bromo-4′-hydroxyacetophenone in an amountcorresponding to 10 mmol is used, the mixed solution of methanol/watermay be used in an amount of about 40 to 100 mL. The amount of sodiumhydrogencarbonate is not specifically limited as long as it is veryexcessive relative to the above-described precipitates as reactionsubstrates. For example, when the reaction is effected under theabove-described conditions, the amount of an aqueous saturated solutionof sodium hydrogencarbonate to be added to the reaction solution can beabout 50 mL.

Here, 2-bromoethanol and t-butyldiphenylchlorosilane (TBDPSCl) arereacted with each other to prepare1-bromo-2-(t-butyldiphenylsiloxy)ethane (FIG. 2-1, Step 3), separately.In this instance, the reaction can be carried out in accordance withordinary methods, for example, the method described in a literature,Organic Syntheses, Coll. Vol. 10, p. 170 (2004); Vol. 79, p. 59 (2002)).

Then, the 2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine preparedabove is sufficiently dried, dissolved in N,N-dimethylformamide, andpotassium carbonate and 1-bromo-2-(t-butyldiphenylsiloxy)ethane wereadded thereto. After this mixture was stirred at about 90° C. for about2 hours, a saturated sodium chloride solution was added, extracted withethyl acetate, concentrated an ethyl acetate layer, and chromatogrampurification is performed to obtain2-[4′-(2″-t-butyldiphenylsiloxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(FIG. 2-1, Step 4). The amount of potassium carbonate to be used may bean amount that can neutralize hydrobromic acid generated from1-bromo-2-(t-butyldiphenylsiloxy)ethane during the reaction, and istypically double to triple the other reactant1-bromo-2-(t-butyldiphenylsiloxy)ethane in molar ratio. Further, the1-bromo-2-(t-butyldiphenylsiloxy)ethane can be used in an excessiveamount relative to the reaction substrate, and is typically about 1.5times the reaction substrate2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine in molar ratio.

Then, t-butyldiphenylsilyl group of the obtained2-[4′-(2″-t-butyldiphenylsiloxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridineis deprotected using tetrabutylammonium fluoride to obtain2-[4′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (FIG. 2-1,Step 5). In this instance, the reaction can be carried out in accordancewith ordinary methods, for example, the method described in aliterature, Organic Syntheses, Coll. Vol. 9, p. 417 (1998); Vol. 74, p.248 (1997)).

The obtained2-[4′-(2″-hydroxyethoxy)]phenyl]-6-iodoimidazo[1,2-a]pyridine wasdissolved in dioxane, and triethylamine is added thereto. Then,bis(tributyltin) and a catalytic amount of tetrakis-triphenylphosphinepalladium are added thereto. This reaction mixture is heated at about90° C. and reacted for about 24 hours, and then a solvent is distilledoff and chromatographic purification is performed to obtain6-tributylstannyl-2-[4′-(2″-hydroxyethoxy)]phenyl]imidazo[1,2-a]pyridineas the target compound (FIG. 2-2, Step 1). The amount ofbis(tributyltin) to be used in this instance may be an amount satisfyinga condition where it is excessive relative to the reaction substrate,specifically, it is about 1.5 times in molar ratio relative to thereaction substrate2-[4′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine.

When a compound with a substituent at the 6-position in theimidazopyridine ring being a trialkylstannyl substituent other than thetributylstannyl substituent is obtained, various bis(trialkyltin)s thatfit purposes can be used instead of bis(tributyltin) in FIG. 2-2,Step 1. For example, when a compound having a trimethylstannylsubstituent as a substituent at the 6-position is synthesized, areaction similar to the above can be performed in FIG. 2-2, Step 1 usingbis(trimethyltin).

A compound with an imidazopyridine ring in which the binding site forthe functional group is a carbon atom other than the carbon at6-position can be obtained by using a compound with a pyridine ring towhich iodine is bonded at a different site instead of2-amino-5-iodopyridine used in FIG. 2-1, Step 2. For example, when abinding site for the functional group is the carbon at 8-position in theimidazopyridine ring, 2-amino-3-iodopyridine may be used instead of2-amino-5-iodopyridine in FIG. 2-1, Step 2.

(A Method for Synthesis of a Radioactive Halogen-labeled Compound)

Next, a method for production of a radioactive halogen-labeled compoundaccording to another aspect of the present invention will be described,taking the case of radioactive iodine-labeled compounds.

The synthesis of radioactive iodine-labeled compounds can be performedby dissolving the labeling precursor compound prepared as aboveprocedure in an inert organic solvent, adding a [¹²³I]sodium iodidesolution obtained by known methods thereto, adding an acid and anoxidizing agent thereto to effect reaction. As an inert organic solventdissolving the labeling precursor compound, various solvents having noreactivity with the labeling precursor, [¹²³I]sodium iodide and the likecan be used, and preferably methanol can be used.

As the acid, may be used various ones, and preferably hydrochloric acid.

The oxidizing agent is not particularly limited as long as it can effectthe oxidation of iodine in the reaction solution, and is preferablyhydrogen peroxide or peracetic acid. The amount of the oxidizing agentto be added may be an amount sufficient to oxidize iodine in thereaction solution.

A compound labeled with a radioactive halogen other than iodine can besynthesized by labeling a labeling precursor that fits a purpose ofsynthesis with a radioactive halogen that fits the purpose. For example,in order to synthesize6-[¹⁸F]fluoro-2-[4′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine, thelabeling precursor2-[4′-(2″-hydroxyethoxy)phenyl]-6-nitroimidazo[1,2-a]pyridine can bereacted with [¹⁸F]fluoride ion in the presence of a phase transfercatalyst and potassium carbonate.

(Methods for Preparing and Using a Diagnostic Agent in Accordance withthe Present Invention)

The diagnostic agent according to the present invention can be preparedas a solution which comprises the present radioactive halogen-labeledcompound blended in water, a physiological saline solution or a Ringer'ssolution optionally adjusted to an appropriate pH, like othercommonly-known radioactive diagnostic agents. In this instance,concentration of the present compound should be adjusted to not morethan the concentration at which stability of the present compound isensured. Dosage of the present compound is not specifically limited aslong as it is sufficient to obtain an image of distribution of anadministered agent. For example, in case of iodine-123 (¹²³I)-labeledcompounds and fluorine-18 (¹⁸F)-labeled compounds, about 50 to 600 MBqper adult body of 60 kg weight can be administered intravenously orlocally. Distribution of administered agents can be imaged by knownmethods. For example, iodine-123 (¹²³I)-labeled compounds can be imagedby a SPECT apparatus while fluorine-18 (¹⁸F)-labeled compounds can beimaged by a PET apparatus.

EXAMPLE

Hereinafter, the present invention is described below in more detail byway of Examples, Comparative Examples and Reference Examples. However,these Examples never limit the scope of the present invention.

Example I

In the following Examples, the names of the individual compounds used inthe experiment are defined as shown in Table 1-1.

TABLE 1-1 Compound name Common name Compound 6-bromo-2-[4′-(3″- I-1fluoropropoxy)phenyl]imidazo[1,2-a]pyridine Compound2-[4′-(3″-fluoropropoxy)phenyl]-6- I-2 iodoimidazo[1,2-a]pyridineCompound 6-bromo-2-[4′-(2″- I-3fluoroethoxy)phenyl]imidazo[1,2-a]pyridine Compound2-[4′-(2″-fluoroethoxy)phenyl]-6-iodoimidazo[1,2- I-4 a]pyridineCompound 2-[4′-(3″-fluoropropoxy)phenyl]-6- I-5iodoimidazo[1,2-a]pyrimidine Compound 2-[4′-(3″-fluoropropoxy)phenyl]-6-I-6 [¹²⁵I]iodoimidazo[1,2-a]pyridine Compound2-[4′-(3″-fluoropropoxy)phenyl]-6- I-7 [¹²³I]iodoimidazo[1,2-a]pyridineCompound 6-bromo-2-[4′-(3″- I-8[¹⁸F]fluoropropoxy)phenyl]imidazo[1,2-a]pyridine Compound2-[4′-(2″-fluoroethoxy)phenyl]-6- I-9 [¹²³I]iodoimidazo[1,2-a]pyridine

Example I-1 Synthesis of6-tributylstannyl-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 1-1, Step 1).

2.15 g (corresponding to 10.0 mmol) of 2-bromo-4′-hydroxyacetophenoneand 1.74 g (corresponding to 10.0 mmol) of 2-amino-5-bromopyridine weredissolved in 50 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 105° C. for 6 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 20 mL of water and 20 mLof methanol. Then, about 25 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 2.41 g (corresponding to8.32 mmol) of 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine (FIG.1-1, Step 2).

290 mg (corresponding to 1.0 mmol) of6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine that was sufficientlydried to remove moisture was dissolved in 10 mL ofN,N-dimethylformamide, and 413 mg (corresponding to 3.0 mmol) ofpotassium carbonate was added thereto. The mixture was supplemented with138 μL (corresponding to 1.5 mmol) of 1-bromo-3-fluoropropane, and thenwas stirred at room temperature for 20.5 hours. After the completion ofthe reaction, the reaction solution was poured into water and extractedthree times with chloroform. The combined chloroform layer was washedwith a saturated sodium chloride solution, dried over anhydrous sodiumsulfate, filtered and concentrated. The resulting crude product waspurified by recycle preparative HPLC (HPLC apparatus: LC-908 (undertrade name; manufactured by Japan Analytical Industry Co., Ltd.);column: two JAIGEL 2H (under trade name; manufactured by JapanAnalytical Industry Co., Ltd.) connected together; mobile phase:chloroform), to obtain 302 mg (corresponding to 0.866 mmol) of6-bromo-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine (FIG. 1-1,Step 3).

85 mg (corresponding to 0.24 mmol) of6-bromo-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine wasdissolved in 10 mL of dioxane, and 2 mL of triethylamine was addedthereto. Then, 185 μL (corresponding to 0.36 mmol) of bis(tributyltin)and 20 mg (at a catalytic amount) of tetrakis-triphenylphosphinepalladium were added thereto. After the reaction mixture was stirred at90° C. for 24 hours, the solvent was distilled off under reducedpressure. The residue was purified by the preparative TLC (elutionsolvent: hexane/ethyl acetate=6/4). Further, the resulting crude productwas purified by recycle preparative HPLC (HPLC apparatus: LC-908 (undertrade name: manufactured by Japan Analytical Industry Co., Ltd.);column: two JAIGEL 2H (under trade name; manufactured by JapanAnalytical Industry Co., Ltd.) connected together; mobile phase:chloroform), to obtain 42 mg (corresponding to 74.2 μmol) of6-tributylstannyl-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine(FIG. 1-1, Step 4).

The NMR measurement results of the resulting6-tributylstannyl-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine(internal standard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ8.01-7.93 (m, 1H), 7.91-7.87 (m, 2H), 7.75-7.74 (m, 1H), 7.63-7.58 (m,1H), 7.20-7.11 (m, 1H), 7.00-6.95 (m, 2H), 4.67 (dt, J_(HF)=47.0 Hz,J=6.0 Hz, 2H) 4.15 (t, J=6.0 Hz, 2H), 2.20 (dquint, J_(HF)=26.1 Hz,J=6.0 Hz, 2H), 1.64-1.47 (m, 6H), 1.39-1.31 (m, 6H), 1.19-1.04 (m, 6H),0.91 (t, J=7.2 Hz, 9H)

Example I-2 Synthesis of2-[4′-(3″-fluoropropoxy)phenyl]-6-[¹²⁵I]iodoimidazo[1,2-a]pyridine

To 53 μL of a solution of6-tributylstannyl-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridinein methanol (concentration: 1 mg/mL), 100 μL of 1 mol/L hydrochloricacid, [¹²⁵I]sodium iodide of 11.1 MBq (20 μL in volume) and 10 μL of 10%(w/v) hydrogen peroxide were added. After the mixed solution was left tostand at room temperature for 10 minutes, the solution was subjected toHPLC under the following conditions, to obtain2-[4′-(3″-fluoropropoxy)phenyl]-6-[¹²⁵I]iodoimidazo[1,2-a]pyridinefraction.

HPLC conditions:

-   Column: Phenomenex Luna C18 (trade name; manufactured by Phenomenex    Co.; size: 4.6×150 mm)-   Mobile phase: 0.1% trifluoroacetic acid/acetonitrile=20/80 to 0/100    (17 minutes, linear gradient)-   Flow rate: 1.0 mL/min.-   Detector: Ultraviolet visible absorptiometer (Detection wavelength:    282 nm) and radioactivity counter (manufactured by raytest: type    STEFFI)

10 ml of water was added to the fraction. The resulting solution waspassed through a reversed phase column (trade name: Sep-Pak (registeredtrademark) Light C18 Cartridges manufactured by Waters: the packedamount of the packing agent: 130 mg) so that the column adsorbs andcollects2-[4′-(3″-fluoropropoxy)phenyl]-6-[¹²⁵I]iodoimidazo[1,2-a]pyridine. Thecolumn was rinsed with 1 mL of water, and then 1 mL of ethanol waspassed therethrough to elute2-[4′-(3″-fluoropropoxy)phenyl]-6-[¹²⁵I]iodoimidazo[1,2-a]pyridine. Theamount of radioactivity of the obtained compound was 5.5 MBq (at the endof synthesis). Further, the TLC analysis was conducted under thefollowing conditions, and as a result, the radiochemical purity of thecompound was 96.0%.

TLC analysis conditions:

-   TLC plate: RP-18F254 (trade name; manufactured by Merck & Co., Inc.)-   Mobile phase: methanol/water=20/1-   Detector: Bio-imaging Analyzer, BAS-2500 (type: BAS-2500    manufactured by FUJIFILM Corporation)

Example I-3 Synthesis of2-[4′-(3″-fluoropropoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine

To 70 μL of a solution of6-tributylstannyl-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridinein methanol (concentration: 1 mg/mL), 100 μL of 1 mol/L hydrochloricacid, [¹²³I]sodium iodide of 260-330 MBq (30-60 μL in volume), 20 μL of1 mmol/L sodium iodide solution and 20 μL of 10% (w/v) hydrogen peroxidewere added. After the mixed solution was heated at 50° C. for 10minutes, the solution was subjected to HPLC under the same conditions asin Example I-2, to obtain2-[4′-(3″-fluoropropoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine as afraction.

10 ml of water was added to the fraction. The resulting solution waspassed through a reversed phase column (trade name: Sep-Pak (registeredtrademark) Light C18 Cartridges manufactured by Waters: the packedamount of the packing agent: 130 mg) so that the column adsorbs andcollects2-[4′-(3″-fluoropropoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine. Thecolumn was rinsed with 1 mL of water, and then 1 mL of ethanol waspassed therethrough to elute2-[4′-(3″-fluoropropoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine. Theamount of radioactivity of the obtained compound was 112-153 MBq at theend of synthesis. Further, the TLC analysis was conducted under the sameconditions as in Example I-2, and as a result, the radiochemical purityof the compound was 97.0%.

Example I-4 Synthesis of6-bromo-2-[4′-(3″-p-toluenesulfonyloxypropoxy)phenyl]imidazo[1,2-a]pyridine

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 1-2, Step 1).

2.15 g (corresponding to 10.0 mmol) of 2-bromo-4′-hydroxyacetophenoneand 1.74 g (corresponding to 10.0 mmol) of 2-amino-5-bromopyridine weredissolved in 50 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 105° C. for 6 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 20 mL of water and 20 mLof methanol. Then, about 25 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 2.41 g (corresponding to8.32 mmol) of 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine (FIG.1-2, Step 2).

1.45 g (corresponding to 5.0 mmol) of6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine that was sufficientlydried to remove moisture was dissolved in 50 mL ofN,N-dimethylformamide, and 2.07 g (corresponding to 15.0 mol) ofpotassium carbonate was added thereto. The mixture was supplemented with680 μL (corresponding to 7.5 mmol) of 3-bromo-1-propanol, and then thesolution was stirred at room temperature for 17 hours. After thecompletion of the reaction, the reaction solution was poured into waterand extracted three times with chloroform. The combined chloroform layerwas washed with a saturated sodium chloride solution, dried overanhydrous sodium sulfate, filtered and concentrated. The resulting crudeproduct was recrystallized from methanol to obtain 1.28 g (correspondingto 3.67 mmol) of6-bromo-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine (FIG.1-2, Step 3).

177 mg (corresponding to 0.5 mmol) of6-bromo-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine wasdissolved in 10 mL of pyridine, and 197 mg (corresponding to 1.0 mmol)of p-toluenesulfonylchloride was added under an ice bath. After thereaction solution was stirred at room temperature for 16 hours, it waspoured into water and extracted three times with chloroform. Thecombined chloroform layer was washed with a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered andconcentrated. The resulting crude product was purified by recyclepreparative HPLC (HPLC apparatus: LC-908 (under trade name: manufacturedby Japan Analytical Industry Co., Ltd.); column: two JAIGEL 2H (undertrade name; manufactured by Japan Analytical Industry Co., Ltd.)connected together; mobile phase: chloroform), to obtain 87 mg(corresponding to 0.17 mmol) of6-bromo-2-[4′-(3″-p-toluenesulfonyloxypropoxy)phenyl]imidazo[1,2-a]pyridine(FIG. 1-2, Step 4).

The NMR measurement results of the resulting6-bromo-2-[4′-(3″-p-toluenesulfonyloxypropoxy)phenyl]imidazo[1,2-a]pyridine(internal standard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ8.26-8.24 (m, 1H), 7.84-7.80 (m, 2H), 7.77-7.74 (m, 2H), 7.74 (s, 1H),7.50 (d, J=9.7 Hz, 1H), 7.26-7.23 (m, 2H), 7.21 (dd, J=9.7, 2.0 Hz, 1H),6.84-6.80 (m, 2H), 4.26 (t, J=6.0 Hz, 2H), 3.98 (t, J=6.0 Hz, 2H), 2.35(s, 3H), 2.13 (quint., J=6.0 Hz, 2H).

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 125 MHz): δ158.67, 146.53, 144.79, 144.08, 132.77, 129.80, 127.87, 127.81, 127.28,126.20, 125.43, 117.87, 114.63, 107.40, 106.76, 66.97, 63.08, 28.85,21.60.

Example I-5 Synthesis of6-bromo-2-[4′-(3″-[¹⁸F]fluoropropoxy)phenyl]imidazo[1,2-a]pyridine

[¹⁸F]fluoride ion-containing H₂ ¹⁸O (radioactivity: 4210 MBq, a valueconverted at the beginning of synthesis) was passed through a Sep-PakLight QMA (under trade name; manufactured by Waters) to adsorb andcollect [¹⁸F]fluoride ions. Then, a potassium carbonate solution (66.7mmol/L, 0.3 mL) and 1.5 mL of a solution of 20 mg (corresponding to 53.2μmol) of Kryptofix 222 (under trade name; manufactured by Merck Co.,Ltd.) in acetonitrile were passed through the column to elute the[¹⁸F]fluoride ions.

The eluate was heated under helium gas stream to 100° C. to evaporatewater, and supplemented with actetonitrile (0.3 mL×2) and azeotropicallydistilled to dryness. To this, 1.0 mL of a solution of 5 mg(corresponding to 10.0 μmol) of6-bromo-2-[4′-(3″-p-toluenesulfonyloxypropoxy)phenyl]imidazo[1,2-a]pyridinesynthesized above in Example I-4 in dimethylformamide was added thereto,and heated at 130° C. for 10 minutes. After the reaction solution wascooled down to 30° C., it was passed through a Sep-Pak Plus Silica(trade name; Waters) each time the reaction solution was supplementedwith ether (3.5 mL×3). The ether solution that had been passed washeated to 60° C. under helium gas stream and concentrated. Theconcentrated solution was diluted with 2 mL of a mixed solution ofmethanol/water/triethylamine=800:200:1.

The resulting solution was purified by HPLC (column: Capcell Pak C18 MG(15 mm i.d.×250 mm, manufactured by Shiseido Co., Ltd.); elutionsolvent: methanol/water/triethylamine=700/300/1). An eluate fractioncontaining the target compound is diluted with 100 mL of water, and thenpassed through a Sep-Pak Plus C18 (trade name, manufactured by Waters)to adsorb and collect the target compound. Then, 20 mL of water waspassed through the column to wash it. Then, 4 mL of ethanol was passedthrough the column to elute a solution of6-bromo-2-[4′-(3″-[¹⁸F]fluoropropoxy)phenyl]imidazo[1,2-a]pyridine inethanol. The obtained radioactivity level was 769 MBq (107 min. afterthe beginning of synthesis). According to the TLC analysis on thefollowing conditions, the radiochemical purity thereof was 95.9%.

TLC analysis conditions:

-   TLC plate: Silica Gel 60 F₂₅₄ (trade name; manufactured by Merck &    Co., Inc.)-   Mobile phase: Chloroform/methanol/triethylamine=500/10/0.5-   Detector: Rita Star (trade name; manufactured by raytest)

Example I-6 Synthesis of6-bromo-2-[4′-(2″-p-toluenesulfonyloxyethoxy)phenyl]imidazo[1,2-a]pyridine

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 1-3, Step 1).

2.15 g (corresponding to 10.0 mmol) of 2-bromo-4′-hydroxyacetophenoneand 1.74 g (corresponding to 10.0 mmol) of 2-amino-5-bromopyridine weredissolved in 50 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 105° C. for 6 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 20 mL of water and 20 mLof methanol. Then, about 25 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 2.41 g (corresponding to8.32 mmol) of 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine (FIG.1-3, Step 2).

621 mg (corresponding to 10.0 mmol) of ethylene glycol was dissolved in100 mL of methylene chloride. To this solution under an ice bath, 3.49 g(corresponding to 15.0 mmol) of silver oxide, 350 mg (corresponding to2.1 mmol) of potassium iodide and 2.10 g (corresponding to 11.0 mmol) ofp-toluenesulfonylchloride were added. The resulting mixture was stirredat 0° C. for 2 hours. Insoluble matters were filtered out of thereaction mixture, and were washed with ethyl acetate. The washings werecombined with the filtrate, and the mixture was concentrated. Theresulting crude product was purified by flash silica gel columnchromatography (elution solvent: hexane/ethyl acetate=1/1) to obtain 643mg (corresponding to 2.97 mmol) of 2-hydroxyethyl-p-toluenesulfonate(FIG. 1-3, Step 3).

To 10 mL of a solution of 639 mg (corresponding to 2.95 mmol) of2-hydroxyethyl-p-toluenesulfonate in tetrahydrofran, 388 mg(corresponding to 1.34 mmol) of6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine and 780 mg(corresponding to 2.97 mmol) of triphenylphosphine were added. Further,6 mL of N,N-dimethylformamide was added thereto to completely dissolvethe contents. To the reaction mixture, 0.58 mL (corresponding to 2.95mmol) of diisopropylazodicarboxylate was added. After the reactionmixture was stirred at room temperature for 17 hours, the reactionsolution was concentrated. The resulting crude product was purified byflash silica gel column chromatography (elution solvent: hexane/ethylacetate=65/35). Insoluble matter in chloroform was filtered out offractions containing a target compound. Further, the resulting crudeproduct was purified by recycle preparative HPLC (HPLC apparatus: LC-908(under trade name; manufactured by Japan Analytical Industry Co., Ltd.);column: two JAIGEL 2H (under trade name; manufactured by JapanAnalytical Industry Co., Ltd.) connected together; mobile phase:chloroform), to obtain 79.7 mg (corresponding to 164 μmol) of6-bromo-2-[4′-(2″-p-toluenesulfonyloxyethoxy)phenyl]imidazo[1,2-a]pyridine(FIG. 1-3, Step 4).

The NMR measurement results of the resulting6-bromo-2-[4′-(2″-p-toluenesulfonyloxyethoxy)phenyl]imidazo[1,2-a]pyridineare shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: dimethylformamide-d7, resonance frequency: 500 MHz): δ8.73-8.71 (m, 1H), 8.19-8.17 (m, 1H), 7.81-7.77 (m, 2H), 7.73-7.70 (m,2H), 7.41-7.38 (m, 1H), 7.39-7.36 (m, 2H), 7.20 (dd, J=9.5, 1.9 Hz),6.85-6.81 (m, 2H), 4.34-4.31 (m, 2H), 4.19-4.15 (m, 2H).

¹³C-NMR (solvent: dimethylformamide-d7, resonance frequency: 125 MHz): δ158.32, 145.91, 145.24, 143.84, 133.15, 130.18, 127.83, 127.54, 127.19,127.15, 126.90, 117.56, 114.86, 108.73, 105.80, 69.28, 65.88, 20.69.

Reference Example I-1 Synthesis of6-bromo-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine(Non-radioactive Fluorinated Form)

As a sample for evaluating affinity with amyloid, solubility in fat andmutagenicity of the present compound, a non-radioactive fluorinated formof 6-bromo-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine wassynthesized.

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 1-4, Step 1).

2.15 g (corresponding to 10.0 mmol) of 2-bromo-4′-hydroxyacetophenoneand 1.74 g (corresponding to 10.0 mmol) of 2-amino-5-bromopyridine weredissolved in 50 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 105° C. for 6 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 20 mL of water and 20 mLof methanol. Then, about 25 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 2.41 g (corresponding to8.32 mmol) of 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine (FIG.1-4, Step 2).

290 mg (corresponding to 1.0 mmol) of6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine that was sufficientlydried to remove moisture was dissolved in 10 mL ofN,N-dimethylformamide, and 413 mg (corresponding to 3.0 mmol) ofpotassium carbonate was added thereto. The mixture was supplemented with138 μL (corresponding to 1.5 mmol) of 1-bromo-3-fluoropropane, and thenwas stirred at room temperature for 20.5 hours. After the completion ofthe reaction, the reaction solution was poured into water and extractedthree times with chloroform. The combined chloroform layer was washedwith a saturated sodium chloride solution, dried over anhydrous sodiumsulfate, filtered and concentrated. The resulting crude product waspurified by recycle preparative HPLC (HPLC apparatus: LC-908 (undertrade name; manufactured by Japan Analytical Industry Co., Ltd.);column: two JAIGEL 2H (under trade name; manufactured by JapanAnalytical Industry Co., Ltd.) connected together; mobile phase:chloroform), to obtain 302 mg (corresponding to 0.866 mmol) of6-bromo-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine (FIG. 1-4,Step 3).

The NMR measurement results of the resulting6-bromo-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine (internalstandard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ 8.23(dd, J=1.9, 0.2 Hz, 1H), 7.88-7.83 (m, 2H), 7.51-7.48 (m, 1H), 8.21 (dd,J=9.5, 1.9 Hz, 1H), 6.99-6.95 (m, 2H), 4.67 (dt, ²J_(HF)=47.1 Hz, J=5.9Hz, 2H), 4.15 (t, J=5.9 Hz, 2H), 2.19 (dquint, ³J_(HF)=25.9 Hz, J=5.9Hz, 2H).

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 125 MHz): δ159.01, 146.61, 144.07, 127.81, 127.38, 126.15, 125.41, 117.87, 114.78,107.41, 106.71, 80.71 (d, ¹J_(CF)=164.6 Hz), 63.59 (d, ³J_(CF)=5.3 Hz),30.43 (d, ²J_(CF)=19.7 Hz).

¹⁹F-NMR (solvent: chloroform-dl, resonance frequency: 470 MHz): δ−222.07 (dd, ²J_(HF)=47.1 Hz, ³J_(HF)=25.9 Hz)

Reference Example I-2 Synthesis of2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(Non-radioactive Fluorinated Form)

As a sample for evaluating affinity with amyloid, solubility in fat andmutagenicity of the present compounds, a non-radioactive fluorinatedform of 2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine wassynthesized.

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 1-5, Step 1).

441 mg (corresponding to 2.0 mmol) of 2-bromo-4′-hydroxyacetophenone and449 mg (corresponding to 2.0 mmol) of 2-amino-5-iodopyridine weredissolved in 15 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 110° C. for 5 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 10 mL of water and 10 mLof methanol. Then, about 10 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 526 mg (corresponding to1.56 mmol) of 2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine (FIG.1-5, Step 2).

673 mg (corresponding to 2.0 mmol) of2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine that was sufficientlydried to remove moisture was dissolved in 25 mL ofN,N-dimethylformamide, and 831 mg (corresponding to 6.0 mmol) ofpotassium carbonate was added thereto. The mixture was supplemented with275 μL (corresponding to 3.0 mmol) of 1-bromo-3-fluoropropane, and thenstirred at room temperature for 24 hours. After the completion of thereaction, the reaction solution was poured into water and extractedthree times with chloroform. The combined chloroform layer was washedwith water and a saturated sodium chloride solution, dried overanhydrous sodium sulfate, filtered and concentrated. The resulting crudeproduct was purified by flash silica gel column chromatography (elutionsolvent: chloroform), and further by recycle preparative HPLC (HPLCapparatus: LC-908 (under trade name; manufactured by Japan AnalyticalIndustry Co., Ltd.); column: two JAIGEL 2H (under trade name;manufactured by Japan Analytical Industry Co., Ltd.) connected together;mobile phase: chloroform), to obtain 349 mg (corresponding to 0.881mmol) of 2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(FIG. 1-5, Step 3).

The NMR measurement results of the resulting2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (internalstandard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ8.37-8.35 (m, 1H), 7.88-7.84 (m, 2H), 7.72 (s, 1H), 7.42-7.39 (m, 1H),7.32 (dd, J=9.4, 1.6 Hz, 1H), 6.99-6.96 (m, 2H), 4.67 (dt, ²J_(HF)=47.0Hz, J=6.0 Hz, 2H), 4.15 (t, J=6.0 Hz, 2H), 2.20 (dquint, ³J_(HF)=25.9Hz, J=6.0 Hz, 2H).

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 125 MHz): δ159.01, 146.23, 144.16, 132.36, 130.28, 127.42, 126.05, 118.31, 114.77,106.90, 80.72 (d, ¹J_(CF)=164.6 Hz), 74.80, 63.57 (d, ³J_(CF)=5.3 Hz),30.42 (d, ²J_(CF)=20.2 Hz).

¹⁹F-NMR (solvent: chloroform-dl, resonance frequency: 470 MHz): δ−222.09 (dd, ²J_(HF)=47.0 Hz, ³J_(HF)=25.9 Hz)

Reference Example I-3 Synthesis of6-bromo-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridine(Non-radioactive Fluorinated Form)

As a sample for evaluating affinity with amyloid, solubility in fat andmutagenicity of the present compounds, a non-radioactive fluorinatedform of 6-bromo-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridine wassynthesized.

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 1-6, Step 1).

2.15 g (corresponding to 10.0 mmol) of 2-bromo-4′-hydroxyacetophenoneand 1.74 g (corresponding to 10.0 mmol) of 2-amino-5-bromopyridine weredissolved in 50 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 105° C. for 6 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 20 mL of water and 20 mLof methanol. Then, about 25 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 2.41 g (corresponding to8.32 mmol) of 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine (FIG.1-6, Step 2).

578 mg (corresponding to 2.0 mmol) of6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine that was sufficientlydried to remove moisture was dissolved in 20 mL ofN,N-dimethylformamide, and 830 mg (corresponding to 6.0 mmol) ofpotassium carbonate was added thereto. The mixture was supplemented with510 μL (corresponding to 3.0 mmol) of 2-fluoroethyl-p-toluenesulfonate,and then the solution was stirred at room temperature for 44.5 hours.After the completion of the reaction, the reaction solution was pouredinto water and extracted three times with chloroform. The combinedchloroform layer was washed with water and a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered andconcentrated. The resulting crude product was purified by flash silicagel column chromatography (elution solvent: chloroform/methanol=100/1),by recycle preparative HPLC (HPLC apparatus: LC-908 (under trade name;manufactured by Japan Analytical Industry Co., Ltd.); column: two JAIGEL2H (under trade name; manufactured by Japan Analytical Industry Co.,Ltd.) connected together; mobile phase: chloroform), and further bypreparative TLC (elution solvent: chloroform/methanol=50:1) to obtain446 mg (corresponding to 1.33 mmol) of6-bromo-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridine (FIG. 1-6,Step 3).

The NMR measurement results of the resulting6-bromo-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridine (internalstandard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ8.23-8.21 (m, 1H), 7.87-7.84 (m, 1H), 7.72 (s, 1H), 7.51-7.47 (m, 1H),7.20 (dd, J=9.5, 1.9 Hz, 1H), 7.01-6.97 (m, 2H), 4.84-4.71 (m, 2H),4.30-4.21 (m, 2H).

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 125 MHz): δ158.62, 146.46, 144.06, 127.85, 127.41, 126.58, 125.42, 117.87, 114.91,107.49, 106.74, 81.86 (d, ¹J_(CF)=170.8 Hz), 67.15 (d, ²J_(CF)=20.2 Hz)

¹⁹F-NMR (solvent: chloroform-dl, resonance frequency: 470 MHz): δ−223.80 (dd, J_(HF)=7.4 Hz, J_(HF)=27.6 Hz)

Reference Example I-4 Synthesis of2-[4′-(2″-fluoroethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(Non-radioactive Fluorinated Form)

As a sample for evaluating affinity with amyloid, solubility in fat andmutagenicity of the present compounds, a non-radioactive fluorinatedform of 2-[4′-(2″-fluoroethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine wassynthesized.

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal.

Then, the resulting solution was filtered and concentrated. Theresulting crude product was purified by flash silica gel columnchromatography (elution solvent: chloroform/methanol=20/1), andrecrystallized from ethyl acetate/petroleum ether, to obtain 7.25 g(corresponding to 33.7 mmol) of 2-bromo-4′-hydroxyacetophenone (FIG.1-7, Step 1).

441 mg (corresponding to 2.0 mmol) of 2-bromo-4′-hydroxyacetophenone and449 mg (corresponding to 2.0 mmol) of 2-amino-5-iodopyridine weredissolved in 15 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 110° C. for 5 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 10 mL of water and 10 mLof methanol. Then, about 10 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 526 mg (corresponding to1.56 mmol) of 2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine (FIG.1-7, Step 2).

368 mg (corresponding to 1.1 mmol) of2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine that was sufficientlydried to remove moisture was dissolved in 15 mL ofN,N-dimethylformamide, and 453 mg (corresponding to 3.3 mmol) ofpotassium carbonate was added thereto. The mixture was supplemented with280 μL (corresponding to 1.6 mmol) of 2-fluoroethyl-p-toluenesulfonate,and then the solution was stirred at room temperature for 22 hours.After the completion of the reaction, the reaction solution was pouredinto water and extracted three times with chloroform. The combinedchloroform layer was washed with water and a saturated sodium chloridesolution, dried over anhydrous sodium sulfate, filtered andconcentrated. The resulting crude product was purified by flash silicagel column chromatography (elution solvent: hexane/ethyl acetate=1/1),and further by recycle preparative HPLC (HPLC apparatus: LC-908 (undertrade name; manufactured by Japan Analytical Industry Co., Ltd.);column: two JAIGEL 2H (under trade name; manufactured by JapanAnalytical Industry Co., Ltd.) connected together; mobile phase:chloroform), to obtain 222 mg (corresponding to 0.580 mmol) of2-[4′-(2″-fluoroethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (FIG. 1-7,Step 3).

The NMR measurement results of the resulting2-[4′-(2″-fluoroethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (internalstandard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ8.35-8.33 (m, 1H), 7.88-7.84 (m, 2H), 7.70 (s, 1H), 7.39 (d, J=9.4 Hz,1H), 7.31 (dd, J=9.4, 1.8 Hz, 1H), 7.01-6.97 (m, 2H), 4.84-4.71 (m, 2H),4.30-4.22 (m, 2H).

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 125 MHz): δ158.62, 146.08, 144.16, 132.38, 130.30, 127.44, 126.52, 118.30, 114.91,106.99, 81.86 (d, ²J_(CF)=170.8 Hz), 74.82, 67.15 (d, ³J_(CF)=20.6 Hz)

¹⁹F-NMR (solvent: chloroform-dl, resonance frequency: 470 MHz): δ−223.74 (dd, ²J_(HF)=47.4 Hz, ³J_(HF)=27.7 Hz)

Reference Example I-5 Synthesis of2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyrimidine(Non-radioactive Fluorinated Form)

As a sample for evaluating affinity with amyloid, solubility in fat andmutagenicity of the present compounds, a non-radioactive fluorinatedform of 2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine wassynthesized.

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 1-8, Step 1).

646 mg (corresponding to 3.0 mmol) of 2-bromo-4′-hydroxyacetophenone and668 mg (corresponding to 3.0 mmol) of 2-amino-5-iodopyrimidine weredissolved in 20 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 110° C. for 8 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 10 mL of water and 10 mLof methanol. Then, about 15 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 3 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 737 mg (corresponding to2.19 mmol) of 2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyrimidine (FIG.1-8, Step 2).

339 mg (corresponding to 1.0 mmol) of2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyrimidine that wassufficiently dried to remove moisture was dissolved in 20 mL ofN,N-dimethylformamide, and 414 mg (corresponding to 3.0 mmol) ofpotassium carbonate was added thereto. The mixture was supplemented with138 μL (corresponding to 1.5 mmol) of 1-bromo-3-fluoropropane, and thenstirred at room temperature for 22 hours. After the completion of thereaction, the reaction solution was poured into water and extractedthree times with chloroform. The combined chloroform layer was washedwith a saturated sodium chloride solution, dried over anhydrous sodiumsulfate, filtered and concentrated. The resulting crude product wasrecrystallized from N,N-dimethylformamide to obtain 236 mg(corresponding to 0.594 mmol) of2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyrimidine (FIG.1-8, Step 3).

The NMR measurement results of the resulting2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyrimidine (internalstandard: dimethylsulfoxide) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: dimethylsulfoxide-d6, resonance frequency: 500 MHz): δ9.27 (d, J=2.3 Hz, 1H), 8.55 (d, J=2.3 Hz, 1H), 8.15 (s, 1H), 7.94-7.90(m, 2H), 7.06-7.02 (m, 2H), 4.62 (dt, ²J_(HF)=47.2 Hz, J=6.1, 2H), 4.14(t, J=6.1 Hz, 2H), 2.13 (dquint, ³J_(HF)=25.5 Hz, J=6.1 Hz, 2H).

¹³C-NMR (solvent: dimethylsulfoxide-d6, resonance frequency: 125 MHz): δ159.16, 154.12, 146.54, 146.26, 139.00, 127.60, 126.06, 115.21, 106.52,81.15 (d, ¹J_(CF)=161.7 Hz), 74.43, 64.07 (d, J_(CF)=5.8 Hz), 30.13 (d,J_(CF)=19.7 Hz).

¹⁹F-NMR (solvent: dimethylsulfoxide-d6, resonance frequency: 470 MHz): δ−220.13 (tt, ²J_(HF)=47.2 Hz, J_(HF)=25.5 Hz).

Reference Example I-6 Synthesis of [¹²⁵I]-IMPY

[¹²⁵I]-IMPY was synthesized in accordance with the following steps foruse in Comparative Examples for evaluation on binding to amyloid

In accordance with the method described in a literature (Zhi-Ping Zhuanget al., J. Med. Chem., 2003, 46, p. 237-243),6-tributylstannyl-2-[4′-(N,N-dimethylamino)phenyl]imidazo[1,2-a]pyridinewas synthesized, and dissolved in methanol (concentration: 1 mg/mL). To53 μL of the resulting solution, 75 μL of 1 mol/L hydrochloric acid, 20μL of [¹²⁵I]sodium iodide of 13.5 MBq and 10 μL of 10% (w/v) hydrogenperoxide were added. After the mixed solution was left to stand at 50°C. for 10 minutes, the solution was subjected to HPLC under the samecondition as described in Example I-2, to obtain [¹²⁵I]-IMPY fraction.

10 ml of water was added to the fraction. The resulting solution waspassed through a reverse phase column (trade name: Sep-Pak (registeredtrademark) Light C18 Cartridges manufactured by Waters; the packedamount of the packing agent: 130 mg), so that the column adsorbs andcollects the [125]-IMPY. The column was rinsed with 1 mL of water, andthen 1 mL of ethanol was passed therethrough, to elute [¹²⁵I]-IMPY. Theobtained radioactivity was 2.6 MBq at the end of synthesis. Further, theTLC analysis was conducted under the same conditions as described inExample I-2, and as a result, the radiochemical purity of the compoundwas 98.0%.

Reference Example I-7 Synthesis of [¹²³I]-IMPY

[¹²³I]-IMPY was prepared in accordance with the following steps for usein Comparative Examples for evaluations on logP_(octanol) andaccumulation in brain.

In accordance with the method described in a literature (Zhi-Ping Zhuanget al., J. Med. Chem., 2003, 46, p. 237-243),6-tributylstannyl-2-[4′-(N,N-dimethylamino)phenyl]imidazo[1,2-a]pyridinewas synthesized, and dissolved in methanol (concentration: 1 mg/mL). To53 μL of the resulting solution, 100 μL of 1 mol/L hydrochloric acid,20-50 μL of [¹²³I]sodium iodide of 190-240 MBq, 10 μL of 1 mmol/L sodiumiodide and 10 μL of 10% (w/v) hydrogen peroxide were added. After themixed solution was left to stand at 50° C. for 10 minutes, the solutionwas subjected to HPLC under the same condition as described in ExampleI-2, to obtain [¹²³I]-IMPY fraction.

10 ml of water was added to the fraction. The resulting solution waspassed through a reverse phase column (trade name: Sep-Pak (registeredtrademark) Light C18 Cartridges manufactured by Waters; the packedamount of the packing agent: 130 mg), so that the column adsorbs andcollects the [¹²³]-IMPY. The column was rinsed with 1 mL of water, andthen 1 mL of ethanol was passed therethrough, to elute [¹²³I]-IMPY. Theobtained radioactivity was 47-56 MBq at the end of synthesis. Further,the TLC analysis was conducted under the same conditions as described inExample I-2, and as a result, the radiochemical purity of the compoundwas 98.0%.

Reference Example I-8 Synthesis of2-(4′-hydroxyphenyl)-6-[¹²⁵I]iodoimidazo[1,2-a]pyridine

In order to prepare calculation formulas for the calculation oflogP_(HPLC), 2-(4′-hydroxyphenyl)-6-[¹²⁵I]iodoimidazo[1,2-a]pyridine wassynthesized in accordance with the following steps.

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 1-9, Step 1).

2.15 g (corresponding to 10.0 mmol) of 2-bromo-4′-hydroxyacetophenoneand 1.74 g (corresponding to 10.0 mmol) of 2-amino-5-bromopyridine weredissolved in 50 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 105° C. for 6 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 20 mL of water and 20 mLof methanol. Then, about 25 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 2.41 g (corresponding to8.32 mmol) of 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine (FIG.1-9, Step 2).

138 mg (corresponding to 0.476 mmol) of6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine was dissolved in 20mL of dioxane, and 2 mL of triethylamine was added thereto. Then, 360 μL(corresponding to 0.713 mmol) of bis(tributyltin) and 20 mg (a catalyticamount) of tetrakis-triphenylphosphine palladium were added thereto.After the reaction mixture was stirred at 90° C. for 22 hours, thesolvent was distilled off under reduced pressure. The residue waspurified by preparative TLC (elution solvent: hexane/ethyl acetate=1/4).Further, the resulting crude product was purified by recycle preparativeHPLC (HPLC apparatus: LC-908 (under trade name: manufactured by JapanAnalytical Industry Co., Ltd.); column: two JAIGEL 2H (under trade name;manufactured by Japan Analytical Industry Co., Ltd.) connected together;mobile phase: chloroform), to obtain 47 mg (corresponding to 94.9 μmol)of 6-tributylstannyl-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine (FIG.1-9, Step 3).

To 53 μL of a solution of6-tributylstannyl-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine in methanol(concentration: 1 mg/mL), 75 μL of 1 mol/L hydrochloric acid, 40 μL of[125]sodium iodide of 136 MBq and 10 μL of 10% (w/v) hydrogen peroxidewere added. After the mixed solution was left to stand at 50° C. for 10minutes, the solution was subjected to HPLC under the same condition asin Example I-2, to obtain2-(4′-hydroxyphenyl)-6-[¹²⁵I]iodoimidazo[1,2-a]pyridine fraction (FIG.1-9, Step 4).

10 ml of water was added to the fraction. The resulting solution waspassed through a reversed phase column (trade name: Sep-Pak (registeredtrademark) Light C18 Cartridges manufactured by Waters: the packedamount of the packing agent: 130 mg) so that the column adsorbs andcollects 2-(4′-hydroxyphenyl)-6-[125I]iodoimidazo[1,2-a]pyridine. Thecolumn was rinsed with 1 mL of water, and then 1 mL of ethanol waspassed therethrough, to elute2-(4′-hydroxyphenyl)-6-[¹²⁵I]iodoimidazo[1,2-a]pyridine. The obtainedradioactivity was 37.5 MBq at the end of synthesis. Further, the TLCanalysis was conducted under the same conditions as in Example I-2, andas a result, the radiochemical purity of the compound was 96.5%.

Reference Example I-9 Synthesis of2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 1-10, Step 1).

441 mg (corresponding to 2.0 mmol) of 2-bromo-4′-hydroxyacetophenone and449 mg (corresponding to 2.0 mmol) of 2-amino-5-iodopyridine weredissolved in 15 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 110° C. for 5 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 10 mL of water and 10 mLof methanol. Then, about 10 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 526 mg (corresponding to1.56 mmol) of 2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine (FIG.1-10, Step 2). The NMR measurement results of the resulting2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine (internal standard:dimethylsulfoxide) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: dimethylsulfoxide-d6, resonance frequency: 500 MHz): δ8.86-8.84 (m, 1H), 8.14 (s, 1H), 7.78-7.74 (m, 2H), 7.40-7.35 (m, 2H),6.86-6.82 (m, 2H).

¹³C-NMR (solvent: dimethylsulfoxide-d6, resonance frequency: 125 MHz): δ158.08, 145.87, 143.87, 132.48, 131.72, 127.67, 124.99, 118.14, 116.14,108.02, 75.85.

Examples I-7 and Comparative Examples I-1 Measurement of Binding toAmyloid

Affinity of the present compounds with amyloid was examined by thefollowing in vitro binding tests.

(1) Aβ₁₋₄₀ (Peptide Institute, INC.) was dissolved in phosphate buffer(pH 7.4) and shaken at 37° C. for 72 hours, to obtain a 1 mg/mLsuspension (hereinafter referred to as amyloid suspension in theseExamples) of aggregated Aβ (hereinafter referred to as amyloid in thisExample).

(2) According to the method described in a literature (Naiki, H., etal., Laboratory Investigation 74, p. 374-383 (1996)), the amyloidsuspension was subjected to qualitative experiment based on fluorescencespectrophotometric method using Thioflavin T (manufactured by Fluka) toconfirm that the aggregated Aβ obtained in (1) was amyloid (measurementconditions: excitation wavelength of 446 nm, and emission wavelength of490 nm).

(3) A solution in ethanol of the compound I-6 synthesized by a methoddescribed above in Example I-2 (radioactive concentration: 37 MBq/mL)was prepared, and diluted with a 0.1% bovine serum albumin-containingphosphate buffer (pH 7.4) to prepare a solution corresponding to 2nmol/L as a total amount of2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine.

(4) To each well of a 96-well microplate, 50 μL of a solution (400 μM ata final concentration) prepared above in (3) and 50 μL of a solution(amyloid concentration may be adjusted in accordance with amyloidconcentration in a sample solution) where amyloid suspension wasdissolved in a 0.1% bovine serum albumin-containing phosphate buffer (pH7.4), were added, and then 150 μL of the same buffer was added toprepare a solution of amyloid at final concentrations of 2.5, 12.5, 25,62.5, 125, 250, 625, and 1000 nmol/L.

(5) The microplate was shaken at a given rate (400 rpm) at 22° C. for 3hours. Then, a mixed solution of each well was filtered through a glassfiber filter (trade name: Mulutiscreen™-FC, manufactured by Millipore),to separate the Compound I-6 attached to amyloid from the free CompoundI-6.

(6) The glass fiber filter used for filtration of the mixed solution waswashed with a 0.1% bovine serum albumin-containing phosphate buffer (0.5mL×5), and then radioactivity of the glass fiber filter was measuredwith an autowell gamma system (manufactured by Aloka, Type: ARC-301B).

(7) A relation between the amount of the Compound I-6 binding to amyloidand the amount of added amyloid was evaluated from the measurementresults of (6). Unspecific binding was determined using a sample towhich the Compound I-2 (non-RI labeled compound) was added to become 100nM (at a final concentration) above in (4) (Example I-7).

(8) Using [¹²⁵I]-IMPY synthesized in the above Reference Example I-6,the same procedure as the above (2) to (6) were carried out to obtaincontrol data (Comparative Example I-1).

A relation between the concentration of amyloid in the sample solutionand the radioactive count on the glass fiber filter measured above in(6) is shown in FIG. 1-11. The radioactivity on the glass fiber wasincreased proportionally to the concentration (addition amount) ofamyloid (Example I-7). On the conditions of the present experiment,amyloid and the compound attached to amyloid are retained in the glassfiber. Therefore, the radioactive count on the glass fiber is a valuereflecting the amount of the compound attached to amyloid, and the slopeof the graph which plotted the radioactive count relative to theconcentration of amyloid is a value which can be an index representingthe intensity of binding to amyloid. Since the value of the radioactivecount of Compound I-6 on the glass fiber was increased with increase ofthe concentration of amyloid, it has been suggested that Compound I-6 isa compound having a property of binding to amyloid. The slope of theline for Compound I-6 was greater than the slope for [¹²⁵I]-IMPY in thesame plot, and thus it has been suggested that the affinity of CompoundI-6 with amyloid is stronger than [¹²⁵I]-IMPY which is known to exhibita strong affinity with amyloid.

The above-mentioned results have implied that Compound I-6 is highlycapable of binding to amyloid.

Examples I-8 to I-12, Comparative Examples I-2 to I-6 Measurement ofAffinity with Amyloid

Affinity of the present compounds with amyloid was examined by thefollowing in vitro binding tests.

(1) Aβ₁₋₄₀ (Peptide Institute, INC.) was dissolved in phosphate buffer(pH 7.4) and shaken at 37° C. for 62-72 hours, to obtain a 1 mg/mLsuspension of aggregated Aβ (hereinafter referred to as amyloidsuspension in this Example).

(2) According to the method described in a literature (Naiki, H., etal., Laboratory Investigation 74, p. 374-383 (1996)), the amyloidsuspension was subjected to qualitative experiment based on fluorescencespectrophotometric method using Thioflavin T (manufactured by Fluka) toconfirm that the aggregated Aβ obtained in (1) was amyloid (measurementconditions: excitation wavelength of 446 nm, and emission wavelength of490 nm).

(3) According to the method described in a literature (Wang, Y., et al.,J. Labeled Compounds Radiopharmaceut. 44, S239 (2001)),[¹²⁵I]2-(3′-iodo-4′-aminophenyl)benzothiazole (hereinafter referred toas [¹²⁵I]3′-1-BTA-0) was prepared from a labeling precursor2-(4′-aminophenyl)benzothiazole, and dissolved in ethanol. As Congo Red,Thioflavin T and 6-methyl-2-[4′-(N,N-dimethylamino)phenyl]benzothiazole(hereinafter referred to as 6-Me-BTA-2), commercially available reagentswere weighed and used as they were.

(4) 2-(3′-Iodo-4′-aminophenyl)benzothiazole (hereinafter referred to as3′-1-BTA-0) and IMPY were synthesized according to the methods describedin a literature (Wang. Y., et al., J. Labelled CompoundsRadiopharmaceut. 44, S239 (2001)) and a literature (Zhuang, Z. P., etal., J. Med. Chem. 46, 237 (2003)) respectively.

(5) Samples in which [¹²⁵I]3′-1-BTA-0, each compound for evaluation andamyloid were dissolved in a 0.1% bovine serum albumin-containingphosphate buffer (pH 7.4) at final concentrations shown in Table 1-2were prepared. The resulting samples were filled in each well (about 0.3mL in volume) of a 96-well microplate.

TABLE 1-2 Final concentrations of each compound in sample solutionsConcentration Compound of compound [¹²⁵I]3′-I- for for BTA-0 Experimentevaluation evaluation concentration Amyloid Comparative 3′-I-BTA-0 Each400 pmol/L 1 μmol/L Example I-2 concentration Comparative Congo Red of0, 0.001, Example I-3 0.01, 0.1, 1, Comparative Thioflavin T 10, 100,and Example I-4 1000 nmol/L Comparative 6-Me-BTA-2 Example I-5Comparative IMPY Example I-6 Example Compound I-8 I-1 Example CompoundI-9 I-2 Example Compound I-10 I-3 Example Compound I-11 I-4 ExampleCompound I-12 I-5

(6) The microplate filled with the sample solutions was shaken at agiven rate (400 rpm) at 22° C. for 3 hours. Then, each sample solutionwas filtered through a glass fiber filter (trade name: Mulutiscreen™-FC,manufactured by Millipore), to separate the [¹²⁵I]3′-I-BTA-0 attached toamyloid from the free [¹²⁵I]3′-I-BTA-0.

(7) The glass fiber filter used for the filtration of each samplesolution was washed with a 0.1% bovine serum albumin-containingphosphate buffer (pH 7.4) (0.5 mL×5), and radioactivity of the glassfiber filter was measured with an autowell gamma system (manufactured byAloka, Type: ARC-301B). The radioactivity was used as the radioactivitylevel of each sample solution for calculating an inhibition ratio(hereinafter, A denotes the radioactivity level in a sample with zero(0) concentration of each compound for evaluation, and B denotes theradioactivity level in a sample with 0.001 nmol/L or higherconcentration of each compound for evaluation).

(8) Separately, a solution of containing 15 μmol/L of 6-Me-BTA-2, 400μmol/L of [¹²⁵I]3′-1-BTA-0 and 1 μmol/L of Aβ₁₋₄₀ were prepared andsubjected to the same procedures as described above in (6) and (7) tomeasure a radioactivity level. The measured radioactivity level wasdefined as the background radioactivity level, and used in thecalculation of the inhibition ratio (hereinafter referred to as BG).

(9) Using the radioactivity levels measured above in (7) and (8), theinhibition ratio was determined by the following formula (I-1).

$\begin{matrix}{\frac{B - {BG}}{A - {BG}} \times 100(\%)} & ( {1\text{-}1} )\end{matrix}$

A graph in which values converted by probit transformation from theobtained inhibition ratios were plotted relative to logarithms ofconcentrations of compounds for evaluation was prepared to obtain anapproximate straight line by the least square method. Using the line,the concentration of each compound for evaluation was determined, atwhich the radioactivity level is half of the level of the sample freefrom each compound for evaluation, and was defined as a 50% inhibitionconcentration of each compound (hereinafter referred to as IC₅₀% value).Using the value as an indicator, affinity of each compound forevaluation with amyloid (aggregated Aβ₁₋₄₀) was evaluated.

IC₅₀% value of each compound for evaluation is shown in Table 1-3.Compounds I-1 to I-5 all showed IC₅₀% values of less than 100 and hadhigher affinity with amyloid (aggregated Aβ₁₋₄₀) than Congo Red andThioflavin T. The results show that Compounds I-1 to I-5 have goodaffinity with amyloid (aggregated Aβ₁₋₄₀). In particular, Compounds I-1to I-4 had higher affinity with amyloid (aggregated Aβ₁₋₄₀) than3′-1-BTA-0 and 6-Me-BTA-2 and had the affinity comparable to IMPY.

TABLE 1-3 IC_(50%) values of the present compounds Compound for IC_(50%)values Experiment evaluation (nmol/L) Comparative Example 3′-I-BTA-010.1 I-2 Comparative Example Congo Red >1000 I-3 Comparative ExampleThioflavin T >1000 I-4 Comparative Example 6-Me-BTA-2 25.4 I-5Comparative Example IMPY 0.8 I-6 Example I-8 Compound I-1 1.4 ExampleI-9 Compound I-2 0.9 Example I-10 Compound I-3 1.8 Example I-11 CompoundI-4 0.9 Example I-12 Compound I-5 55.4

Example I-13 to I-14, Comparative Example I-7 Measurement of PartitionCoefficient Based on the Octanol Extraction Method

Partition coefficients based on the octanol extraction method(hereinafter referred to as logP_(octanol)) were measured, which aregenerally known as an indicator of permeability of compounds through theblood-brain barrier (hereinafter referred to as BBB).

To 2 mL of octanol, 10 μL of a solution containing Compound I-7 (ExampleI-13) and Compound I-8 (Example I-14) and 2 mL of 10 mmol/L phosphatebuffer (pH 7.4) were added, and stirred for 30 seconds. After themixture was centrifuged with a low-speed centrifuge (2000 rpm×60 min.),the octanol layer and the water layer were sampled each in an amount of1 mL, and subjected to measurement of radioactivity count with anautowell gamma system (manufactured by Aloka, Type: ARC-301B). Using theobtained radioactivity count, logP_(octanol) was calculated inaccordance with the equation (1-2).

$\begin{matrix}{{\log\; P_{octanol}} = {\log_{10}( \frac{{Radioactivity}\mspace{14mu}{count}\mspace{14mu}{of}\mspace{14mu}{octanol}\mspace{14mu}{layer}}{{Radioactivity}\mspace{14mu}{count}\mspace{14mu}{of}\mspace{14mu}{water}\mspace{14mu}{layer}} )}} & ( {1\text{-}2} )\end{matrix}$

The results are shown in Table 1-4. Both Compounds I-7 and I-8 showedlogP_(octanol) values between 1 and 3. It is known that compoundspermeable to BBB show a logP_(octanol) value between 1 and 3 (Douglas D.Dischino et al., J. Nucl. Med., (1983), 24, p. 1030-1038). Thus, it isimplied that both compounds have a BBB permeability comparable to IMPY.

TABLE 1-4 logP_(octanol) value of the present compound ExperimentCompound logP_(octanol) value Comparative [¹²³I]-IMPY 2.1 Example I-7Example I-13 Compound I-7 2.1 Example I-14 Compound I-8 2.0

Example I-15 to I-19, Comparative Example I-8 Measurements of PartitionCoefficient Based on HPLC

The partition coefficient by HPLC (hereinafter referred to aslogP_(HPLC)) was measured by the following method. It is known that thelogP_(HPLC) shows the same numerical value at a pH of 7.2 to 7.4 as thelogP_(octanol) value which is generally known as an indicator ofpermeability of compounds to BBB (Franco Lombardo et al., J. Med. Chem.,(2000), 43, p. 2922-2927).

First, compounds for evaluation shown in Table 1-5 were dissolved at aconcentration of 1 mg/mL in methanol containing 10% dimethylsulfoxide toprepare sample solutions. One μL of the sample solution was subjected toHPLC analysis under the following conditions to determine the elutiontime (t₀) of the solvent and the elution time (t_(R)) of each compound.

TABLE 1-5 Compounds for evaluation in experiments Compound forExperiment evaluation Comparative Example IMPY I-8 Example I-15 CompoundI-1 Example I-16 Compound I-2 Example I-17 Compound I-3 Example I-18Compound I-4 Example I-19 Compound I-5

HPLC conditions:

-   Column: Prodigy ODS (3) (product name; manufactured by phenomenex;    size: 4.6×250 mm)-   Mobile phase: a mixed solution of 50 mM triethylamine phosphate (pH    7.2)/acetonitrile=40/60-   Flow rate: 0.7 mL/min.-   Detector: ultraviolet visible absorptiometer (detection wavelength:    282 nm)

Using the obtained t₀ and t_(R), the retention factor (hereinafterreferred to as K′_(HPLC) value) of each compound for evaluation wasdetermined according to the following calculation formula (I-3).K′ _(HPLC)=(t _(R) −t ₀)/t ₀  (1-3)

Separately, 10 μL each of a solution of2-(4′-hydroxyphenyl)-6-[¹²⁵I]iodoimidazo[1,2-a]pyridine (37 MBq/mL inradioactivity concentration) synthesized above in Reference I-8 and asolution of Compound I-6 (37 MBq/mL in radioactivity concentration) wasadded to 2 mL of octanol prepared separately, and 2 mL of 10 mmol/Lphosphate buffer (pH 7.4) was further added to the respective solutions.After the individual solutions were stirred for 30 seconds, thesolutions were centrifuged at 2,000 rpm for 60 minutes. 1 mL each of theoctanol phase and the water phase was fractionated, and theradioactivity was measured by an autowell gamma system (manufactured byAloka Co., Ltd.: Type ARC-301B). Based on the obtained radioactivity,logP_(octanol) values were calculated according to the above equation(1-2).

Further, a solution of Compound I-2 and2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine prepared above inReference I-9 were each subjected to HPLC analysis in the same manner asdescribed above to determine K′_(HPLC) values in respective compounds.

A graph was prepared, in which the logP_(octanol) values of Compound I-6and 2-(4′-hydroxyphenyl)-6-[¹²⁵I]iodoimidazo[1,2-a]pyridine arerespectively plotted relative to the log₁₀K′_(HPLC) values of CompoundI-2 and 2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine, so that theslope and the intercept on the axis Y of the straight line weredetermined. Using these values, the following formula (I-4) wasdetermined, provided that the logP_(octanol) value is equal to thelogP_(HPLC) value at a pH of 7.2 to 7.4.log P _(HPLC)=0.96(log₁₀ K′ _(HPLC))+1.59  (1-4)

Using K′_(HPLC) obtained for each compound for evaluation, thelogP_(HPLC) value of each compound for evaluation was determinedaccording to the above calculation formula (I-4).

The results are shown in Table 1-6. As shown in the Table, thelogP_(HPLC) values of Compounds I-1 through I-5 were all between 1 and3. As mentioned above, it is known that compounds permeable to BBB havea logP_(octanol) value between 1 and 3 (Douglas D. Dischino et al., J.Nucl. Med., (1983), 24, p. 1030-1038). Further, as mentioned above, itis known that the logP_(HPLC) shows the same value at a pH of 7.2 to 7.4as the logP_(octanol) (Franco Lombardo et al., J. Med. Chem., (2000),43, p. 2922-2927). The above-mentioned results imply that Compounds I-1to I-5 have a BBB-permeable property.

TABLE 1-6 logP_(HPLC) value of the present compound Experiment CompoundlogP_(HPLC) value Comparative Example IMPY 2.1 I-8 Example I-15 CompoundI-1 2.0 Example I-16 Compound I-2 2.1 Example I-17 Compound I-3 1.9Example I-18 Compound I-4 1.9 Example I-19 Compound I-5 1.8

Example I-20 to I-21, Comparative Example I-9 Measurement ofTransferability into Brain and Clearance

Using Compound I-7 (Example I-20) and Compound I-8 (Example I-21), atime course change of radioactive accumulation in brain of male Wistarrats (7-week old) was measured.

0.05 mL (20-30 MBq/mL in radioactive concentration) of a solution ofCompound I-7 (Example I-20) in a 10 mg/mL ascorbic acid-containingphysiological saline solution, a solution of Compound I-8 (Example I-21)in a 10 mg/mL ascorbic acid-containing physiological saline solution anda solution of [¹²³I]-IMPY (Comparative Example I-9) prepared above inReference Example I-7 in a 10 mg/mL ascorbic acid-containingphysiological saline solution were each injected under thiopentalanesthesia into the tail vein of the rats. The rats were sacrificed bybleeding from abdominal artery, and brains were removed and subjected tomeasurement of radioactivity (hereinafter referred to as A in thisExample) with an autowell gamma system (Type: ARC-301B manufactured byAloka Co., Ltd.) and further subjected to measurement of mass of brains2, 5, 30 and 60 minutes after injection. Also, radioactivity(hereinafter referred to as B in this Example) of 0.05 mL of a 1000-folddiluted solution of the injected solution was measured in the samemanner as above. Using these measurement results, radioactivedistribution per unit weight of brain (% ID/g) at the respective timepoints was calculated in accordance with the following formula (I-5).

Three animals were used for Experiment I-20 and Comparative ExperimentI-9, and two animals were used for Experiment I-21 at the respectivetime points.

$\begin{matrix}{{\%\mspace{14mu}{{ID}/g}} = {\frac{A}{B \times 1000 \times {brain}\mspace{14mu}{weight}} \times 100}} & ( {1\text{-}5} )\end{matrix}$

The results are shown in Table 1-7. As shown in Table 1-7, Compounds I-7and I-8 showed an accumulation higher than [¹²³I]-IMPY at the time pointof two minutes after the injection, and then showed a tendency torapidly clear away in 60 minutes. These results suggest that CompoundsI-7 and I-8 possess excellent transferability to brain and rapidclearance from brain comparable to [¹²³I]-IMPY.

TABLE 1-7 Radioactive distribution in brain of the present compoundafter intravenous injection (rats) Radioactive distribution per unitweight (% ID/g) Compound After 2 min. After 5 min. After 30 min. After60 min. Example Compound 1.10 0.75 0.12 0.05 I-20 I-7 Example Compound1.20 0.75 0.18 0.12 I-21 I-8 Comparative ¹²³I-IMPY 1.02 0.99 0.20 0.08Example I-9

Example I-22 Confirmation of Imaging of Amyloid in Brain

The following experiment was carried out in order to examine whetheramyloid in brain can be imaged by the compound of the present invention.

(1) Aβ₁₋₄₀ (manufactured by Peptide Institute, INC.) was dissolved inphosphate buffer (pH 7.4) and shaken at 37° C. for 72 hours, to obtain 1mg/mL of a suspension of aggregated Aβ (hereinafter referred to asamyloid suspension in this Example).

(2) 25 μL (corresponding to 25 μg) of the amyloid suspension wasinjected into an amygdaloid nucleus on one side of a male Wistar rat(7-week old). As a control, 25 μL of a phosphate buffered physiologicalsaline solution (pH 7.4) was injected into an amygdaloid nucleus on theother side of the rat. The rats were examined 1 day after the injectionof the amyloid suspension and the phosphate buffered physiologicalsaline solution (pH 7.4).

(3) Compound I-7 was dissolved in a 10 mg/mL ascorbic acid-containingphysiological saline solution to obtain a sample solution (32 MBq/mL inradioactivity concentration). This solution was injected into the ratthrough the tail vein (dosage: 0.5 mL, dosed radioactivity: 16 MBqequivalent).

(4) Brain was removed 60 minutes after the injection to prepare a brainslice of 10 μm in thickness with a microtome (type: CM3050S,manufactured by LEICA). The brain slice was exposed to an imaging platefor 20 hours, and then image analysis was carried out by use of aBio-imaging Analyzer (type: BAS-2500; manufactured by FUJIFILMCorporation).

(5) After the completion of the image analysis using the Bio-imagingAnalyzer, pathological staining with Thioflavin T was carried out toperform imaging by use of a fluorescence microscope (manufactured byNIKON Corporation; type: TE2000-U model; excitation wavelength: 400-440nm; detection wavelength: 470 nm). Thus, it was confirmed that amyloidwas deposited on the slice (FIG. 1-12 b).

FIG. 1-12 shows images by autoradiogram and Thioflavin T staining of thebrain slice of the rat to which amyloid was injected intracerebrally. Asshown in this figure, a marked accumulation of radioactivity wasobserved in the amygdaloid nucleus on the side to which the amyloidsuspension was injected. From the result of Thioflavin T staining in thesite where radioactivity is accumulated, it was confirmed that amyloidwas present in the accumulation site. On the other hand, no significantaccumulation of radioactivity was observed in the amygdaloid nucleus onthe side to which the physiological saline solution was injected,compared with the other sites.

These results suggest that Compound I-7 possesses a property ofaccumulating on intracerebral amyloid and a capability of imagingintracerebral amyloid.

Example I-23 to I-26 Reverse Mutation Test

In order to examine mutagenicity of Compound I-1, I-2, I-4 and I-5,reverse mutation test using Salmonella typhimurium TA98 and TA100(hereinafter referred to as Ames test) was conducted.

The test was conducted without addition of S9mix and with addition ofS9mix. Dimethylsulfoxide was used as a negative control. A positivecontrol was 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide in case S9mix wasnot added, and 2-aminoanthracene in case S9mix was added.

The amount of each sample to be added to the test plate was 7 dosages(geometric ratio 4) with the maximum dose being 1250 μg/plate forCompounds I-1 and I-5, and 7 dosages (geometric ratio 3) with themaximum dose being 5000 μg/plate for Compounds I-2 and I-4. After asample to be examined and a strain (TA98 or TA100), or a sample to beexamined, S9mix and the strain were mixed together, the mixture wasmultilayered using soft agar on a medium of a test plate, and thenincubated at 37° C. for 48 hours. Judgment was made by counting thenumber of reverse mutation colonies on the plate after the incubation,and when the number of reverse mutation colonies was not less than twotimes the number in negative control and showed concentration-dependentincrease, mutagenicity was determined to be positive.

The results are shown in Table 1-8. The numbers of reverse mutationcolonies of the respective strains in the group treated with CompoundsI-1, I-2, I-4 and I-5 were less than two times the number in the grouptreated with the negative control, regardless of addition of S9mix andthe addition amount of a sample to be examined. From the aforementionedresults, it is judged that Compounds I-1, I-2, I-4 and I-5 are negativein the Ames test and have no mutagenicity.

TABLE 1-8 Results of Ames test Mutagenicity Without addition Withaddition of of S9mix S9mix Compound TA98 TA100 TA98 TA100 ExampleCompound Negative Negative Negative Negative I-23 I-1 Example CompoundNegative Negative Negative Negative I-24 I-2 Example Compound NegativeNegative Negative Negative I-25 I-4 Example Compound Negative NegativeNegative Negative I-26 I-5

Example I-27 Synthesis of6-tributylstannyl-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridine

88 mg (corresponding to 0.260 mmol) of6-bromo-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridine obtained inReference Example I-3 was dissolved in 10.0 mL of dioxane, and 2.0 mL oftriethylamine was added thereto. Then, 0.20 mL (corresponding to 0.39mmol) of bis(tributyltin) and 20.1 mg (a catalytic amount) oftetrakis-triphenylphosphine palladium were added thereto. After thereaction mixture was stirred at 90° C. for 9 hours, the solvent wasdistilled off under reduced pressure. The residue was purified by flashsilica gel column chromatography (elution solvent: hexane/ethylacetate=4/1) to obtain 71.6 mg (corresponding to 0.131 mmol) of6-tributylstannyl-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridine(FIG. 1-13, Step 1).

The NMR measurement results of the obtained6-tributylstannyl-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridine(internal standard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl; resonance frequency: 500 MHz): δ 7.97(s, 1H), 7.90 (d, J=8.7 Hz, 2H), 7.58 (d, J=8.7 Hz, 1H), 7.14 (d, J=8.7Hz, 1H), 6.99 (d, J=8.7 Hz, 2H), 4.77, (dt, J=47.2, 4.1 Hz, 2H), 3.99(dt, J=28.0, 4.1 Hz, 2H), 1.59-1.53 (m, 6H), 1.39-1.32 (m, 6H),1.13-1.10 (m, 6H), 0.92 (t, J=7.3 Hz, 9H).

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ 158.3,145.6, 144.9, 131.2, 130.0, 127.4, 121.9, 116.9, 114.9, 106.4, 82.6,81.3, 67.2, 29.0, 27.3, 13.6, 9.8.

Example I-28 Synthesis of2-[4′-(2″-fluoroethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine

To 35 μL of a solution of6-tributylstannyl-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridinein methanol (concentration: 1 mg/mL), 100 μL of 1 mol/L hydrochloricacid, [¹²³I]sodium iodide of 614 MBq (100 μL in volume), 10 μL of 1mol/L sodium iodide solution and 20 μL of 10% (w/v) hydrogen peroxidewere added. After the mixed solution was heated at 50° C. for 10minutes, the solution was subjected to HPLC under the same conditions asin Example I-2, to obtain2-[4′-(2″-fluoroethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridinefraction.

10 ml of water was added to the fraction. The resulting solution waspassed through a reversed phase column (trade name: Sep-Pak (registeredtrademark) Light C8 Cartridges manufactured by Waters: the packed amountof the packing agent: 130 mg) so that the column adsorbs and collects2-[4′-(2″-fluoroethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine. Thecolumn was rinsed with 1 mL of water, and then 1 mL of ethanol waspassed therethrough to elute2-[4′-(2″-fluoroethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine. Theamount of radioactivity of the obtained compound was 64 MBq at the endof synthesis. Further, the TLC analysis was conducted under thefollowing conditions, and as a result, the radiochemical purity of thecompound was 97.0%.

TLC analysis conditions:

-   TLC plate: Silica Gel 60 F₂₅₄ (trade name; manufactured by Merck &    Co., Inc.)-   Mobile phase: Chloroform/methanol/triethylamine=100/1/2-   Detector: Rita Star (trade name; manufactured by raytest)

Example I-29, Comparative Example I-10 Measurement of PartitionCoefficient Based on the Octanol Extraction Method

A diethyl ether solution of Compound I-9 prepared in Example I-28(Example I-29) and a diethyl ether solution of [¹²³I]-IMPY (ComparativeExample I-10) were each diluted with a 10 mg/mL ascorbic acid-containingphysiological saline solution, and adjusted radioactive concentration tobe 20-30 MBq/mL. To 2 mL of octanol, 10 μL each of the prepared samplesolutions was added, and 2 mL of a 10 mmol/L phosphate buffer (pH 7.4)was further added, followed by stirring for 30 seconds. After themixture was centrifuged with a low-speed centrifuge (2000 rpm×60 min.),the octanol layer and the water layer were sampled each in an amount of1 mL, and subjected to measurement of radioactivity count with anautowell gamma system (Type: ARC-301B, manufactured by Aloka). Using theobtained radioactivity count, logP_(octanol) was calculated inaccordance with the equation (1-6).

$\begin{matrix}{{\log\; P_{octanol}} = {\log_{10}( \frac{{Radioactivity}\mspace{14mu}{count}\mspace{14mu}{of}\mspace{14mu}{octanol}\mspace{14mu}{layer}}{{Radioactivity}\mspace{14mu}{count}\mspace{14mu}{of}\mspace{14mu}{water}\mspace{14mu}{layer}} )}} & ( {1\text{-}6} )\end{matrix}$

The results are shown in Table 1-9. Compound I-9 also showed alogP_(octanol) value between 1 and 3. It is known that compoundspermeable to BBB show a logP_(octanol) value between 1 and 3 (Douglas D.Dischino et al., J. Nucl. Med., (1983), 24, p. 1030-1038). Thus, it isimplied that Compound I-9 has a BBB-permeability comparable to IMPY.

TABLE I-9 logP_(octanol) value of the present compound ExperimentCompound logP_(octanol) value Comparative [¹²³I]-IMPY 2.1 Example I-10Example I-29 Compound I-9 2.1

Example I-30, Comparative Example I-11 Measurement of Transferabilityinto Brain and Clearance (2)

Using Compound I-9, a time course change of radioactive accumulation inbrain of male Wistar rats (7-week old) was measured.

A solution of Compound I-9 (Example I-30) and a solution of [¹²³I]-IMPY(Comparative Example I-11) prepared above in Reference Example each in a10 mg/mL ascorbic acid-containing physiological saline solution (20-31MBq/mL in radioactive concentration) were prepared. 0.05 mL each ofthese solutions was injected under thiopental anesthesia into the tailvein of respective Wistar rats (7-week old). The rats were sacrificed bybleeding from abdominal artery, and brains were removed and subjected tomeasurement of mass of brains and further subjected to measurement ofradioactivity (hereinafter referred to as A in this Example) with asingle channel analyzer (detector type: SP-20 manufactured by OHYO KOKENKOGYO Co., Ltd.) 2, 5, 30 and 60 minutes after the injection. Also,radioactivity (hereinafter referred to as B in this Example) of the restof the whole body was measured in the same manner as above. Using thesemeasurement results, radioactive distribution per unit weight of brain(% ID/g) at the respective time points were calculated in accordancewith the following formula (I-7).

Three animals were used for experiment at the respective time points.

$\begin{matrix}{{\%\mspace{14mu}{{ID}/g}} = {\frac{A}{B \times {brain}\mspace{14mu}{weight}} \times 100}} & ( {1\text{-}7} )\end{matrix}$

The results are shown in Table 1-10. As shown in Table 1-10, CompoundI-9 showed a significant radioactive accumulation like [¹²³I]-IMPY atthe time point of two minutes after the injection, and then showed atendency to rapidly clear away in 60 minutes. These results suggest thatCompound I-9 possesses excellent transferability to brain and rapidclearance from brain like [¹²³I]-IMPY.

TABLE 1-10 Radioactive distribution in brain of the present compoundafter intravenous injection (rats) Radioactive distribution per unitweight (% ID/g) Compound After 2 min. After 5 min. After 30 min. After60 min. Example Compound 0.72 0.49 0.07 0.02 I-30 I-9 Comparative¹²³I-IMPY 1.19 0.97 0.23 0.09 Example I-11

Example I-31 Confirmation of Imaging of Amyloid in Brain

(1) Aβ₁₋₄₂ (Wako) was dissolved in phosphate buffer (pH 7.4) and shakenat 37° C. for 72 hours, to obtain a 1 mg/mL suspension of aggregated Aβ(hereinafter referred to as amyloid suspension in this Example).

(2) 2.5 μL (corresponding to 25 μg) of the amyloid suspension wasinjected into an amygdaloid nucleus on one side of a male Wistar rat(7-week old). As a control, 2.5 μL of a phosphate buffered physiologicalsaline solution (pH 7.4) was injected into an amygdaloid nucleus on theother side of the rat. The rats were examined 1 day after the injectionof the amyloid suspension and the phosphate buffered physiologicalsaline solution (pH 7.4).

(3) Compound I-9 was dissolved in a 10 mg/mL ascorbic acid-containingphysiological saline solution to obtain a sample solution (21 MBq/mL inradioactivity concentration in a sample solution, Example I-31). Thissolution was injected under thiopental anesthesia into the rat throughthe tail vein (dosage: 0.5 mL, dosed radioactivity: 11-15 MBqequivalent).

(4) Brain was removed 60 minutes after the injection to prepare a brainslice of 10 μm in thickness with a microtome (type: CM3050S,manufactured by LEICA). The brain slice was exposed to an imaging platefor 20 hours, and then image analysis was carried out by use of aBio-imaging Analyzer (type: BAS-2500; manufactured by FUJIFILMCorporation).

(5) After the completion of the image analysis using the Bio-imagingAnalyzer, pathological staining with Thioflavin T was carried out toperform imaging by use of a fluorescence microscope (manufactured byNIKON Corporation; type: TE2000-U model; excitation wavelength: 400-440nm; detection wavelength: 470 nm). Thus, it was confirmed that amyloidwas deposited on the slice (FIG. 1-14).

FIG. 1-14 shows images by autoradiogram and Thioflavin T staining of thebrain slice of the rat to which amyloid was injected intracerebrally. Asshown in this figure, a marked accumulation of radioactivity in thespecimen to which Compound I-9 was injected was also observed in theamygdaloid nucleus on the side to which the amyloid suspension wasinjected. On the other hand, no significant accumulation ofradioactivity was observed in the amygdaloid nucleus on the side towhich the physiological saline solution was injected, compared with theother sites. On the autoradiogram, little accumulation of radioactivitywas observed at sites other than the sites to which amyloid wasinjected. From the result of Thioflavin T staining, it was confirmedthat amyloid was present in the site where radioactivity is accumulated(FIG. 1-14). These results imply that Compound I-9 possesses a propertyof accumulating on intracerebral amyloid and a capability of imagingintracerebral amyloid.

Example I-32 Chromosome Aberration Test

In order to examine whether Compound I-4 can induce chromosomeaberration, the chromosome aberration test was conducted using ChineseHamster fibroblast cell line (CHL/IU cell) in a culture system with orwithout addition of S9 by short-term treatment process and in a 24-hourculture system by continuous treatment process. The addition amount of asample to be tested was set to 1.2, 0.6, 0.3, and 0.15 mg/mL for all therespective culture systems.

When appearance frequency of cells with chromosome aberration apparentlyincreased as compared to negative control group and dose dependency wasobserved, or when the appearance frequency apparently increased at asingle dose and reproducibility was observed, positive determination wasgiven, and otherwise negative determination was given.

As the results of the test, the appearance frequency of cells havingstructural aberration or numerical aberration (diploid) in all theculture systems treated with Compound I-4 was comparable to that of thenegative control group. On the other hand, the positive control groupfor the respective culture systems showed a marked increase of theappearance frequency of cells having structural aberration. From theabove results, the capability of inducing chromosome aberration ofCompound I-4 was judged to be negative under the test conditions.

Example I-33 Micronucleus Test

In order to study mutagenicity (in vivo) of Compound I-4, induction ofmicronucleated polychromatic erythrocyte (hereinafter referred to asMNPCE) was examined using bone marrow cells of Crlj:CD1(ICR) male mouse.

0 mg/kg (negative control group), 250, 500, 1000 and 2000 mg/kg (testsample group) were set as doses for the test. Mice were sacrificed 24and 48 hours after single oral administration, and bone marrow smear wasprepared and observed. Further, a single dose of MMC at 2 mg/kg wasabdominally administrated in positive control group, mice weresacrificed 24 hours after the administration, and then bone marrow smearwas prepared and observed.

When appearance frequency of MNPCE in each administration group showed adose-dependent increase or a statistically significant increase ascompared to negative control group, positive determination was given,and otherwise negative determination was given. As statistical analysis,significance tests were conducted by Wilcoxon rank sum test for theappearance frequency of MNPCE and the ratio of polychromatic erythrocyte(hereinafter referred to as PCE) to the total red blood cell(hereinafter referred to as RBC) in each administration group concerningthe test sample and positive control groups relative to the negativecontrol group and concerning these groups relative to each other wherethe significance level was set to less than 5% and less than 1%,respectively.

From the results of the test, no statistically significant differencewas observed on the appearance frequency of MNPCE and the ratio of PCEto RBC for the test sample group as compared to the negative controlgroup. On the other hand, a significant increase was observed on theappearance frequency of MNPCE for the positive control group as comparedto the negative control group. Based on these results, mutagencity (invivo) of the test sample was judged to be negative because no inductionof micronucleus in mouse bone marrow cells was observed under the abovetest conditions using Compound I-4.

Example II

In the following Examples, the names of the individual compounds used inthe experiment are defined as shown in Table 2-1.

TABLE 2-1 Compound name Common name Compound2-[4′-(2″-hydroxyethoxy)phenyl]-6- II-1 [¹²³I]iodoimidazo[1,2-a]pyridineCompound 2-(4′-ethoxyphenyl)-6-[¹²³I]iodoimidazo[1,2- II-2 a]pyridineCompound 2-[4′-(2″-hydroxyethoxy)phenyl]-6- II-3iodoimidazo[1,2-a]pyridine Compound 2-[3′-(2″-hydroxyethoxy)phenyl]-6-II-4 [¹²³I]iodoimidazo[1,2-a]pyridine Compound2-[3′-(2″-hydroxyethoxy)phenyl]-6- II-5 iodoimidazo[1,2-a]pyridineCompound 2-[4′-(3″-hydroxypropoxy)phenyl]-6- II-6[¹²³I]iodoimidazo[1,2-a]pyridine Compound2-[4′-(3″-hydroxypropoxy)phenyl]-6- II-7 iodoimidazo[1,2-a]pyridine

Example II-1 Synthesis of2-[4′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(non-radioactive iodinated form)

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 2-1, Step 1).

441 mg (corresponding to 2.0 mmol) of 2-bromo-4′-hydroxyacetophenone and449 mg (corresponding to 2.0 mmol) of 2-amino-5-iodopyridine weredissolved in 15 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 110° C. for 5 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 10 mL of water and 10 mLof methanol. Then, about 10 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 526 mg (corresponding to1.56 mmol) of 2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine (FIG.2-1, Step 2).

Separately, 2.50 g (corresponding to 20.0 mmol) of 2-bromoethanol and2.72 g (corresponding to 40.0 mmol) of imidazole were dissolved in 10 mLof dimethylformamide (DMF), and cooled to 0° C. Then, 5.50 g(corresponding to 20.0 mmol) of t-butyldiphenylchlorosilane (TBDPSCl)was added thereto. After the reaction mixture was stirred at roomtemperature for 18 hours, a saturated sodium chloride solution wasadded, and extracted three times with ethyl acetate. The combined ethylacetate layers were dried over anhydrous sodium sulfate, andconcentrated under reduced pressure. The resulting crude product waspurified by silica gel column chromatography (elution solvent:hexane/ethyl acetate=10/1) to obtain 7.04 g (corresponding to 19.4 mmol)of 1-bromo-2-(t-butyldiphenylsiloxy)ethane (FIG. 2-1, Step 3).

200 mg (corresponding to 0.595 mmol) of2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine was dissolved in 3.0mL of dimethylformamide, and 247 mg (corresponding to 1.79 mmol) ofpotassium carbonate was added thereto. Then, 259 mg (corresponding to0.714 mmol) of 1-bromo-2-(t-butyldiphenylsiloxy)ethane was addedthereto. After the reaction mixture was stirred at 90° C. for 2 hours, asaturated sodium chloride solution was added, and extracted three timeswith ethyl acetate. The combined ethyl acetate layers were dried overanhydrous sodium sulfate, and concentrated under reduced pressure. Theresulting crude product was purified by silica gel column chromatography(elution solvent: hexane/ethyl acetate=2/1) to obtain 368 mg(corresponding to 0.595 mmol) of2-[4′-(2″-t-butyldiphenylsiloxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(FIG. 2-1, Step 4).

368 mg (corresponding to 0.595 mmol) of2-[4′-(2″-t-butyldiphenylsiloxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridinewas dissolved in 1.0 mL of tetrahydrofuran (THF), and 0.70 mL of a 1.0mol/L tetrahydrofuran solution of tetrabutylammoniumfluoride (TBAF) wasadded thereto. After the reaction mixture was stirred at roomtemperature for 2 hours, ammonium chloride solution was added, followedby addition of 5.0 mL of water and 2.0 mL of acetonitrile. Then,precipitates were filtered. The filtered precipitates were washed withwater and acetonitrile in this order, to obtain 226 mg (corresponding to0.595 mmol) of2-[4′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (FIG. 2-1,Step 5).

The NMR measurement results of the resulting2-[4′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (internalstandard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: dimethylsulfoxide-d6; resonance frequency: 500 MHz): δ8.95 (s, 1H), 8.27 (s, 1H), 7.87 (d, J=8.7 Hz, 2H), 7.54-7.46 (m, 2H),7.04 (d, J=8.7 Hz, 2H), 4.04 (t, J=4.6 Hz, 2H), 3.73 (t, J=4.6 Hz, 2H).

¹³C-NMR (solvent: dimethylsulfoxide-d6, resonance frequency: 500 MHz): δ158.9, 143.0, 142.4, 133.5, 131.5, 127.1, 124.4, 116.7, 114.8, 108.1,76.7, 69.5, 59.4.

Example II-2 Synthesis of6-tributylstannyl-2-[4′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine

100 mg (corresponding to 0.263 mmol) of2-[4′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine obtained inExample II-1 was dissolved in 4.0 mL of dioxane, and 2.0 mL oftriethylamine was added thereto. Then, 0.20 mL (corresponding to 0.39mmol) of bis(tributyltin) and 20.1 mg (a catalytic amount) oftetrakis-triphenylphosphine palladium were added thereto. After thereaction mixture was stirred at 90° C. for 21 hours, the solvent wasdistilled off under reduced pressure. The residue was purified by flashsilica gel column chromatography (elution solvent: hexane/ethylacetate=1/2), to obtain 75.3 mg (corresponding to 0.139 mmol) of6-tributylstannyl-2-[4′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine(FIG. 2-2, Step 1).

The NMR measurement results of the resulting6-tributylstannyl-2-[4′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine(internal standard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl; resonance frequency: 500 MHz): δ 7.98(s, 1H), 7.89 (d, J=8.7 Hz, 1H), 7.75 (s, 1H), 7.56 (d, J=8.7 Hz, 1H),7.15 (d, J=8.7 Hz, 1H), 6.98 (d, J=8.7 Hz, 1H), 4.13 (t, J=4.6 Hz, 2H),3.99 (t, J=4.6 Hz, 2H), 2.63 (s, 3H), 1.64-1.51 (m, 6H), 1.36 (sextet,J=7.3 Hz, 6H), 1.19-1.06 (m, 6H), 0.92 (t, J=7.3 Hz, 9H).

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ 158.6,145.7, 145.0, 131.2, 130.0, 127.4, 127.2, 121.9, 116.9, 114.8, 106.4,69.3, 61.4, 29.0, 27.3, 13.7, 9.8.

Example II-3 Synthesis of2-[4′-(2″-hydroxyethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine

To 60 μL of a mixed solution of6-tributylstannyl-2-[4′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine(concentration: 1 mg/mL) in methanol/dimethylsulfoxide (mixing ratio:9/1), 150 μL of 1 mol/L hydrochloric acid, 15 μL of 1 mmol/L sodiumiodide, 250 μL of [¹²³I]sodium iodide of 274 MBq and 15 μL of 10% (w/v)hydrogen peroxide were added. After the mixed solution was left to standat 50° C. for 10 minutes, it was subjected to HPLC under the followingconditions, to obtain2-[4′-(2″-hydroxyethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridinefraction.

HPLC conditions:

-   Column: Phenomenex Luna C18 (trade name; manufactured by Phenomenex    Co.; size: 4.6×150 mm)-   Mobile phase: 0.1% trifluoroacetic acid/acetonitrile=20/80 to 0/100    (17 minutes)-   Flow rate: 1.0 mL/min.-   Detector: Ultraviolet visible absorptiometer (Detection wavelength:    282 nm) and radioactivity counter (manufactured by raytest: type    STEFFI)

10 ml of water was added to the fraction. The resulting solution waspassed through a reversed phase column (trade name: Sep-Pak (registeredtrademark) Light C8 Cartridges manufactured by Waters: the packed amountof the packing agent: 145 mg) so that the column adsorbs and collects2-[4′-(2″-hydroxyethoxy)phenyl]-6-[123]iodoimidazo[1,2-a]pyridine. Thecolumn was rinsed with 1 mL of water, and then 1 mL of diethyl ether waspassed therethrough to elute2-[4′-(2″-hydroxyethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine. Theamount of radioactivity of the obtained compound was 22 MBq at the endof synthesis. Further, the TLC analysis was conducted under thefollowing conditions, and as a result, the radiochemical purity of thecompound was 97%.

TLC analysis conditions:

-   TLC plate: Silica Gel 60 F₂₅₄ (trade name; manufactured by Merck &    Co., Inc.)-   Mobile phase: Chloroform/methanol/triethylamine=100/1/2-   Detector: Rita Star (trade name; manufactured by raytest)

Example II-4 Synthesis of2-(4′-ethoxyphenyl)-6-iodoimidazo[1,2-a]pyridine (non-radioactiveiodinated form)

30 mL of ethyl acetate was added to 2.72 g (corresponding to 12.2 mmol)of cupric bromide to obtain a suspension, to which 1.00 g (correspondingto 6.09 mmol) of 4′-ethoxyacetophenone was added. Then, the mixture wasrefluxed. After 3 hours, the reaction mixture was cooled down to roomtemperature and filtered. Then, the resulting filtrate was concentratedunder reduced pressure. The residue was dissolved in ethyl acetate andconcentrated. The resulting crude product was purified by silica gelcolumn chromatography (elution solvent: hexane/ethyl acetate=10/1), toobtain 1.20 g (corresponding to 4.94 mmol) of2-bromo-4′-ethoxyacetophenone (FIG. 2-3, Step 1).

1.20 g (corresponding to 4.94 mmol) of 2-bromo-4′-ethoxyacetophenone and1.09 g (corresponding to 4.95 mmol) of 2-amino-5-iodopyridine weredissolved in 20 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 110° C. for 1.5 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered. Then, the precipitates were washed withacetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 10 mL of water and 5 mLof methanol. Then, about 20 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 10 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 1.64 g (corresponding to4.50 mmol) of 2-(4′-ethoxyphenyl)-6-iodoimidazo[1,2-a]pyridine (FIG.2-3, Step 2).

The NMR measurement results of the resulting2-(4′-ethoxyphenyl)-6-iodoimidazo[1,2-a]pyridine (internal standard:tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: dimethylsulfoxide-d6; resonance frequency: 500 MHz): δ9.06 (s, 1H), 8.38 (s, 1H), 7.86 (d, J=8.7 Hz, 2H), 7.77-7.57 (m, 2H),7.06 (d, J=8.7 Hz, 2H), 4.10 (q, J=6.9 Hz, 2H), 1.36 (t, J=6.9 Hz, 3H).

¹³C-NMR (solvent: dimethylsulfoxide-d6, resonance frequency: 500 MHz): δ159.3, 141.1, 140.3, 135.9, 132.0, 127.3, 122.1, 115.3, 114.9, 108.5,78.6, 63.2, 14.5.

Example II-5 Synthesis of6-tributylstannyl-2-(4′-ethoxyphenyl)imidazo[1,2-a]pyridine

364 mg (corresponding to 1.00 mmol) of2-(4′-ethoxyphenyl)-6-iodoimidazo[1,2-a]pyridine obtained in ExampleII-4 was dissolved in 4.0 mL of dioxane, and 2 mL of triethylamine wasadded thereto. Then, 0.76 mL (corresponding to 1.5 mmol) ofbis(tributyltin) and 76.3 mg (a catalytic amount) oftetrakis-triphenylphosphine palladium were added thereto. After thereaction mixture was stirred at 90° C. for 23 hours, the solvent wasdistilled off under reduced pressure. The residue was purified by flashsilica gel column chromatography (elution solvent: hexane/ethylacetate=5/1), to obtain 331 mg (corresponding to 0.628 mmol) of6-tributylstannyl-2-(4′-ethoxyphenyl)imidazo[1,2-a]pyridine (FIG. 2-4,Step 1).

The NMR measurement results of the resulting6-tributylstannyl-2-(4′-ethoxyphenyl)imidazo[1,2-a]pyridine (internalstandard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl; resonance frequency: 500 MHz): δ 7.96(s, 1H), 7.88 (d, J=8.7 Hz, 2H), 7.74 (s, 1H), 7.58 (d, J=8.7 Hz, 1H),7.14 (d, J=8.7 Hz, 1H), 6.96 (d, J=8.7 Hz, 2H), 4.07 (q, J=6.9 Hz, 2H),1.63-1.49 (m, 6H), 1.43 (t, J=6.9 Hz, 3H), 1.39-1.31 (m, 6H), 1.18-1.04(m, 6H), 0.90 (t, J=7.3 Hz, 9H).

¹³C-NMR (solvent: chloroform-dl, resonance frequency: 500 MHz): δ 159.0,145.7, 145.2, 131.2, 130.1, 127.4, 126.7, 121.9, 117.0, 114.8, 106.4,63.6, 29.1, 27.4, 15.0, 13.8, 9.9.

Example II-6 Synthesis of2-(4′-ethoxyphenyl)-6-[¹²³I]iodoimidazo[1,2-a]pyridine

To 60 μL of a mixed solution of6-tributylstannyl-2-(4′-ethoxyphenyl)imidazo[1,2-a]pyridine(concentration: 1 mg/mL) in methanol/dimethylsulfoxide (mixing ratio:9/1), 90 μL of 2 mol/L hydrochloric acid, 15 μL of 1 mmol/L sodiumiodide, 100 μL of [¹²³I]sodium iodide of 436 MBq and 15 μL of 10% (w/v)hydrogen peroxide were added. After the mixed solution was left to standat 50° C. for 10 minutes, it was subjected to HPLC under the followingconditions to obtain2-(4′-ethoxyphenyl)-6-[¹²³I]iodoimidazo[1,2-a]pyridine fraction

HPLC conditions:

-   Column: Phenomenex Luna C18 (trade name; manufactured by Phenomenex    Co.; size: 4.6×150 mm)-   Mobile phase: 0.1% trifluoroacetic acid/acetonitrile=2.0/80 to 0/100    (17 minutes)-   Flow rate: 1.0 mL/min.-   Detector: Ultraviolet visible absorptiometer (Detection wavelength:    282 nm) and radioactivity counter (manufactured by raytest: type    STEFFI)

10 ml of water was added to the fraction. The resulting solution waspassed through a reversed phase column (trade name: Sep-Pak (registeredtrademark) Light C8 Cartridges manufactured by Waters: the packed amountof the packing agent: 145 mg) so that the column adsorbs and collects2-(4′-ethoxyphenyl)-6-[¹²³I]iodoimidazo[1,2-a]pyridine. The column wasrinsed with 1 mL of water, and then 1 mL of diethyl ether was passedtherethrough to elute2-(4′-ethoxyphenyl)-6-[¹²³I]iodoimidazo[1,2-a]pyridine. The amount ofradioactivity of the obtained compound was 88 MBq at the end ofsynthesis. Further, the TLC analysis was conducted under the followingconditions, and as a result, the radiochemical purity of the compoundwas 98%.

TLC analysis conditions:

-   TLC plate: Silica Gel 60 F₂₅₄ (trade name; manufactured by Merck &    Co., Inc.)-   Mobile phase: Chloroform/methanol/triethylamine=100/1/2-   Detector: Rita Star (trade name; manufactured by raytest)

Reference Example II-1 Synthesis of [¹²³I]-IMPY

[¹²³I]-IMPY was synthesized in accordance with the following steps foruse in Comparative Examples for evaluations on measurement oflogP_(octanol) and accumulations in brain.

In accordance with the method described in a literature (Zhi-Ping Zhuanget al., J. Med. Chem., 2003, 46, p. 237-243),6-tributylstannyl-2-[4′-(N,N-dimethylamino)phenyl]imidazo[1,2-a]pyridinewas synthesized, and dissolved in methanol (concentration: 1 mg/mL). To53 μL of the resulting solution, 75 μL of 1 mol/L hydrochloric acid,60-70 μL of [¹²³I]sodium iodide of 224-253 MBq, 10 μL of a 1 mmol/Lsodium iodide solution and 15 μL of 10% (w/v) hydrogen peroxide wereadded. After the mixed solution was left to stand at 50° C. for 10minutes, the solution was subjected to HPLC under the same conditions asin Example II-3, to obtain [¹²³I]-IMPY fraction.

10 ml of water was added to the fraction. The resulting solution waspassed through a reversed phase column (trade name: Sep-Pak (registeredtrademark) Light C8 Cartridges manufactured by Waters; the packed amountof the packing agent: 145 mg), so that the column adsorbs and collectsthe [¹²³]-IMPY. The column was rinsed with 1 mL of water, and then 1 mLof diethyl ether was passed therethrough, to elute [¹²³I]-IMPY. Theobtained radioactivity was 41-57 MBq at the end of synthesis. Further,the TLC analysis was conducted under the same conditions as described inExample II-3, and as a result, the radiochemical purity of the compoundwas 93%.

Example II-7, Comparative Example II-1 to II-3 Measurement of Affinitywith Amyloid

Affinity of the present compounds with amyloid was examined by thefollowing in vitro binding tests.

(1) Aβ₁₋₄₂ (Wako) was dissolved in phosphate buffer (pH 7.4) and shakenat 37° C. for 72 hours, to obtain 1 mg/mL of a suspension (hereinafterreferred to as amyloid suspension in this Example) of aggregated Aβ(hereinafter referred to as amyloid in this Example).

(2) According to the method described in a literature (Naiki, H., etal., Laboratory Investigation 74, p. 374-383 (1996)), the amyloidsuspension was subjected to qualitative experiment based on fluorescencespectrophotometric method using Thioflavin T (manufactured by Fluka) toconfirm that the aggregated Aβ obtained in (1) was amyloid (measurementconditions: excitation wavelength of 446 nm, and emission wavelength of490 nm).

(3) According to the method described in a literature (Wang, Y., et al.,J. Labeled Compounds Radiopharmaceut. 44, S239 (2001)),[¹²⁵I]2-(3′-iodo-4′-aminophenyl)benzothiazole (hereinafter referred toas [¹²⁵I]3′-1-BTA-0) was prepared from a labeling precursor2-(4′-aminophenyl)benzothiazole, and dissolved in ethanol. As Congo Red,Thioflavin T and 6-methyl-2-[4′-(N,N-dimethylamino)phenyl]benzothiazole(hereinafter referred to as 6-Me-BTA-2), commercially available reagentswere weighed and used as they were.

(4) IMPY was synthesized according to the method described in aliterature (Zhuang, Z. P., et al., J. Med. Chem. 46, 237 (2003))

(5) Each compound for evaluation or ethanol solution thereof, an ethanolsolution of [¹²⁵I]3′-1-BTA-0 prepared above in (3) and amyloidsuspension prepared above in (1) were dissolved in 1% bovine serumalbumin-containing phosphate buffer (pH 7.4), and samples at finalconcentrations of each compound for evaluation, [¹²⁵I]3′-1-BTA-0 andamyloid shown in Table 2-2 respectively was prepared.

TABLE 2-2 Final concentrations of each compound in sample solutionsConcentration Compound of compound [¹²⁵I]3′-I- for for BTA-0 Experimentevaluation evaluation concentration Amyloid Comparative Congo Red Each400 pmol/L 1 μmol/L Example concentration II-1 of 0, 0.001, ComparativeThioflavin T 0.01, 0.1, 1, Example 10, 100, 1000 II-2 nmol/L ComparativeIMPY Example II-3 Example Compound II-7 II-3

(6) Each sample solution prepared above in (5) was filled in each well(about 0.3 mL in volume) of a 96-well microplate. The microplate filledwith the sample solutions was shaken at a given rate (400 rpm) at 22° C.for 3 hours. Then, each sample solution was filtered through a glassfiber filter (trade name: Mulutiscreen™-FC, manufactured by Millipore),to separate the [¹²⁵I]3′-1-BTA-0 attached to amyloid from the free[¹²⁵I]3′-1-BTA-0.

(7) The glass fiber filter used for the filtration of each samplesolution was washed with 1% bovine serum albumin-containing phosphatebuffer (pH 7.4) (0.5 mL×5), and radioactivity of the glass fiber filterwas measured with an autowell gamma system (manufactured by Aloka, Type:ARC-301B) (hereinafter, A denotes the radioactivity level in a samplewith zero (0) concentration of each compound for evaluation, and Bdenotes the radioactivity level in a sample with 0.001 nmol/L or higherconcentration of each compound for evaluation).

(8) Separately, a solution containing 15 μmol/L of 6-Me-BTA-2, 400μmol/L of [¹²⁵I]3′-1-BTA-0 and 1 μmol/L of amyloid were prepared andsubjected to the same procedures as described above in (7) and (8) tomeasure a radioactivity level. The measured radioactivity level wasdefined as the background radioactivity level, and used in thecalculation of the inhibition ratio (hereinafter referred to as BG).

(9) Using the radioactivity levels measured above in (7) and (8), theinhibition ratio was determined by the following formula (2-1).

$\begin{matrix}{\frac{B - {BG}}{A - {BG}} \times 100(\%)} & ( {2\text{-}1} )\end{matrix}$

A graph in which values converted by probit transformation from theobtained inhibition ratios were plotted relative to logarithms ofconcentrations of compounds for evaluation was prepared to obtain anapproximate straight line by the least square method. Using the line, a50% inhibition concentration of each compound for evaluation(hereinafter referred to as IC₅₀% value) was determined. Using the valueas an indicator, affinity of each compound for evaluation with amyloidwas evaluated.

IC_(50%) value of each compound for evaluation is shown in Table 2-3.Compounds II-3 showed IC_(50%) values of less than 100 and had higheraffinity with amyloid than Congo Red and Thioflavin T which aregenerally known to have affinity with amyloid. The results show thatCompounds II-3 has good affinity with amyloid like IMPY.

TABLE 2-3 IC_(50%) values of the present compounds Compound for IC_(50%)values Experiment evaluation (nmol/L) Comparative Example CongoRed >1000 II-1 Comparative Example Thioflavin T >1000 II-2 ComparativeExample IMPY 25.8 II-3 Example II-7 Compound II-3 66.9

Example II-8 to II-9, Comparative Example II-4 Measurement of PartitionCoefficient Based on the Octanol Extraction Method

Partition coefficients based on the octanol extraction method(hereinafter referred to as logP_(octanol)) were measured, which aregenerally known as an indicator of permeability of compounds through theblood-brain barrier (hereinafter referred to as BBB).

A diethyl ether solution of Compound II-1 prepared in Example II-3(Example II-8), a diethyl ether solution of Compound II-2 prepared inExample II-6 (Example II-9), and a diethyl ether solution of [¹²³I]-IMPYprepared in Reference Example II-1 (Comparative Example II-2) were eachdiluted with 10 mg/mL ascorbic acid-containing physiological salinesolution, and adjusted to radioactive concentration of 20-30 MBq/mL. To2 mL of octanol, 10 μL each of the prepared sample solutions was added,2 mL of 10 mmol/L phosphate buffer (pH 7.4) was added, followed bystirring for 30 seconds. After the mixture was centrifuged with alow-speed centrifuge (2000 rpm×60 min.), the octanol layer and the waterlayer were sampled each in an amount of 1 mL, and subjected tomeasurement of radioactivity count with an autowell gamma system (Type:ARC-301B, manufactured by Aloka). Using the obtained radioactivitycount, logP_(octanol) was calculated in accordance with the equation(2-2).

$\begin{matrix}{{\log\; P_{octanol}} = {\log_{10}( \frac{{Radioactivity}\mspace{14mu}{count}\mspace{14mu}{of}\mspace{14mu}{octanol}\mspace{14mu}{layer}}{{Radioactivity}\mspace{14mu}{count}\mspace{14mu}{of}\mspace{14mu}{water}\mspace{14mu}{layer}} )}} & ( {2\text{-}2} )\end{matrix}$

The results are shown in Table 2-4. All compounds showed logP_(octanol)value between 1 and 3. It is known that compounds permeable to BBB showa logP_(octanol) value between 1 and 3 (Douglas D. Dischino et al., J.Nucl. Med., (1983), 24, p. 1030-1038). Thus, it is implied that bothcompounds have a BBB permeability like IMPY.

TABLE 2-4 logP_(octanol) value of the present compound ExperimentCompound logP_(octanol) value Comparative [¹²³I]-IMPY 1.9 Example II-4Example II-8 Compound II-1 1.8 Example II-9 Compound II-2 2.1

Example II-10 to II-11, Comparative Example II-5 Measurement ofTransferability into Brain and Clearance

Using Compound II-1 (Example II-10) and Compound II-2 (Example II-11), atime course change of radioactive accumulation in brain of male Wistarrats (7-week old) was measured.

A diethyl ether solution of Compound II-1 (Example II-10) prepared inExample II-3, a diethyl ether solution of Compound II-2 (Example II-11)prepared in Example II-6 and a diethyl ether solution of [¹²³I]-IMPY(Comparative Example II-5) prepared in Reference Example II-1 were eachdiluted with 10 mg/mL ascorbic acid-containing physiological salinesolution to adjust radioactive concentration to 8-12 MBq/mL. 0.05 mLeach of the prepared sample solutions was injected under thiopentalanesthesia into the tail vein of the rats. The rats were sacrificed bybleeding from abdominal artery, and brains were removed and subjected tomeasurement of mass of brains and further subjected to measurement ofradioactivity (hereinafter referred to as A in this Example) with asingle channel analyzer (detector type SP-20 manufactured by OHYO KOKENKOGYO Co., Ltd.) 2, 5, 30 and 60 minutes after the injection. Further,the radioactivity level of the rest of the whole body was measured inthe same manner as above (hereinafter referred to as B in this Example).Using these measurement results, radioactive distribution per unitweight of brain (% ID/g) at the respective time points were calculatedin accordance with the following formula (2-3).

Three animals were used for the experiment at the respective timepoints.

$\begin{matrix}{{\%\mspace{14mu}{{ID}/g}} = {\frac{A}{B \times {brain}\mspace{14mu}{weight}} \times 100}} & ( {2\text{-}3} )\end{matrix}$

The results are shown in Table 2-5. As shown in Table 2-5, CompoundsII-1 and II-2 showed a significant radioactive accumulation like[¹²³I]-IMPY at the time point of two minutes after the injection, andthen showed a tendency to rapidly clear away in 60 minutes. Theseresults suggest that both Compounds II-1 and II-2 possess excellenttransferability to brain and rapid clearance from brain like[¹²³I]-IMPY.

TABLE 2-5 Radioactive distribution in brain of the present compoundafter intravenous injection (rats) Radioactive distribution per unitweight (% ID/g) Compound After 2 min. After 5 min. After 30 min. After60 min. Example Compound 0.90 0.52 0.06 0.01 II-10 II-1 Example Compound0.89 0.66 0.13 0.04 II-11 II-2 Comparative ¹²³I-IMPY 1.19 0.97 0.23 0.09Example II-5

Comparative Example II-6 Ex Vivo Autoradiogram of ¹²³I-IMPY Using Ratsof Amyloid Injected Model

(1) Aβ₁₋₄₀ (manufactured by Peptide Institute, INC.) was dissolved inphosphate buffer (pH 7.4) and shaken at 37° C. for 72 hours, to obtain 1mg/mL of a suspension of aggregated Aβ (hereinafter referred to asamyloid suspension in this Example).

(2) 2.5 μL (corresponding to 25 μg) of the amyloid suspension wasinjected into an amygdaloid nucleus on one side of a male Wistar rat(7-week old). As a control, 2.5 μL of a phosphate buffered physiologicalsaline solution (pH 7.4) was injected into an amygdaloid nucleus on theother side of the rat. The rats were examined 1 day after the injectionof the amyloid suspension and the phosphate buffered physiologicalsaline solution (pH 7.4).

(3) [¹²³I]-IMPY was dissolved in a 10 mg/mL ascorbic acid-containingphysiological saline solution to obtain a sample solution (29 MBq/mL inradioactivity concentration in the sample solution). This solution wasinjected under thiopental anesthesia into the rat through the tail vein(dosage: 0.5 mL, dosed radioactivity: 14.5 MBq equivalent).

(4) Brain was removed 60 minutes after the injection to prepare a brainslice of 10 μm in thickness with a microtome (type: CM3050S,manufactured by LEICA). The brain slice was exposed to an imaging platefor 20 hours, and then image analysis was carried out by use of aBio-imaging Analyzer (type: BAS-2500; manufactured by FUJIFILMCorporation).

(5) After the completion of the image analysis using the Bio-imagingAnalyzer, pathological staining with Thioflavin T was carried out toperform imaging by use of a fluorescence microscope (manufactured byNIKON Corporation; type: TE2000-U model; excitation wavelength: 400-440nm; detection wavelength: 470 nm). Thus, it was confirmed that amyloidwas deposited on the slice (FIG. 2-5 b).

FIG. 2-5 shows images by autoradiogram and Thioflavin T staining of thebrain slice of the rat to which amyloid was injected intracerebrally. Asshown in this figure, a marked accumulation of radioactivity wasobserved in the amygdaloid nucleus on the side to which the amyloidsuspension was injected, but also non-specific accumulation was observedin white matter where amyloid was not injected.

Example II-12 Confirmation of Imaging of Amyloid in Brain

The following experiment was carried out in order to examine whetheramyloid in brain can be imaged by the compound of the present invention.

(1) Aβ₁₋₄₂ (Wako) was dissolved in phosphate buffer (pH 7.4) and shakenat 37° C. for 72 hours, to obtain 1 mg/mL of a suspension of aggregatedAβ (hereinafter referred to as amyloid suspension in the Examples).

(2) 2.5 μL (corresponding to 25 μg) of the amyloid suspension wasinjected into an amygdaloid nucleus on one side of a male Wistar rat(7-week old). As a control, 2.5 μL of a phosphate buffered physiologicalsaline solution (pH 7.4) was injected into an amygdaloid nucleus on theother side of the rat. The rats were examined 1 day after the injectionof the amyloid suspension and the phosphate buffered physiologicalsaline solution (pH 7.4).

(3) Compound II-1 was dissolved in a 10 mg/mL ascorbic acid-containingphysiological saline solution to obtain a sample solution (22 MBq/mL inradioactivity concentration in the sample solution). This solution wasinjected under thiopental anesthesia into the rat through the tail vein(dosage: 0.5 mL, dosed radioactivity: 11-13 MBq equivalent).

(4) Brain was removed 60 minutes after the injection to prepare a brainslice of 10 μm in thickness with a microtome (type: CM3050S,manufactured by LEICA). The brain slice was exposed to an imaging platefor 20 hours, and then image analysis was carried out by use of aBio-imaging Analyzer (type: BAS-2500; manufactured by FUJIFILMCorporation).

(5) After the completion of the image analysis using the Bio-imagingAnalyzer, pathological staining with Thioflavin T was carried out toperform imaging by use of a fluorescence microscope (manufactured byNIKON Corporation; type: TE2000-U model; excitation wavelength: 400-440nm; detection wavelength: 470 nm). Thus, it was confirmed that amyloidwas deposited on the slice (FIG. 2-6 b).

FIG. 2-6 shows images by autoradiogram and Thioflavin T staining of thebrain slice of the rat to which amyloid was injected intracerebrally. Asshown in this figure, a marked accumulation of radioactivity wasobserved in the amygdaloid nucleus on the side to which the amyloidsuspension was injected. On the other hand, no significant accumulationof radioactivity was observed in the amygdaloid nucleus on the side towhich the physiological saline solution was injected, compared with theother sites. On the autoradiogram, little accumulation of radioactivitywas observed at sites other than the sites to which amyloid wasinjected. From the result of Thioflavin T staining, it was confirmedthat amyloid was present in the site where radioactivity is accumulated(FIG. 2-6 b).

Thus, Compound II-1 showed little radioactive accumulation at the sitesother than amyloid injected sites, and showed little non-specificbinding to the white matter observed in [¹²³I]-IMPY. These resultssuggest that Compound II-1 possesses an excellent capability of imagingamyloid in the total autoradiogram image. These results also suggestthat Compound II-1 is a compound that possesses a high specificity toimaging of intracerebral amyloid.

Example II-13 Confirmation of Imaging of Amyloid in Brain

The same procedures as in Example II-12 were performed except using a 10mg/mL solution of Compound II-2 in ascorbic acid (the radioactiveconcentration of the sample solution was 25 MBq/mL).

FIG. 2-7 shows images by autoradiogram and Thioflavin T staining of thebrain slice of the rat to which amyloid was injected intracerebrally. Asshown in this figure, a marked accumulation of radioactivity wasobserved in the amygdaloid nucleus on the side to which the amyloidsuspension was injected. From the result of Thioflavin T staining in thesite where radioactivity was accumulated, it was confirmed that amyloidwas present in the accumulation site. On the other hand, no significantaccumulation of radioactivity was observed in the amygdaloid nucleus onthe side to which the physiological saline solution was injected,compared with the other sites.

Compound II-2 showed some radioactive accumulation in sites other thanamyloid injected sites, but the accumulation was highly suppressed ascompared to ¹²³I-IMPY. As a result, the whole image was provided with ahigh capability of imaging amyloid.

These results suggest that Compound II-2 is a compound that possesses ahigh specificity to imaging of intracerebral amyloid.

Example II-14 Synthesis of2-[3′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(non-radioactive iodinated form)

50 mL of ethyl acetate was added to 8.60 g (corresponding to 46.0 mmol)of cupric bromide to obtain a suspension, to which a solution of 2.50 g(corresponding to 22.0 mmol) of 3′-hydroxyacetophenone in 50 m l ofethyl acetate was added. Then, the resulting mixture was refluxed. After2 hours, the reaction mixture was cooled down to room temperature andfiltered. The resulting filtrate was concentrated under reducedpressure. The residue was dissolved in ethyl acetate and subjected todecoloring operation with addition of active charcoal. Then, theresulting solution was filtered and concentrated. The resulting crudeproduct was purified by flash silica gel column chromatography (elutionsolvent: hexane/ethyl acetate=2/1) to obtain 4.42 g (corresponding to20.6 mmol) of 2-bromo-3′-hydroxyacetophenone (FIG. 2-8, Step 1).

987 mg (corresponding to 4.55 mmol) of 2-bromo-3′-hydroxyacetophenoneand 1.00 g (corresponding to 4.55 mmol) of 2-amino-5-iodopyridine weredissolved in 50 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 110° C. for 2 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 10 mL of water and 1 mLof methanol. Then, about 10 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine.

Precipitates were filtered and recovered from the resulting mixture,sufficiently washed with water, and dried under reduced pressure, toobtain 927 mg (corresponding to 2.76 mmol) of2-(3′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine (FIG. 2-8, Step 2).Separately, 2.50 g (corresponding to 20.0 mmol) of 2-bromoethanol and2.72 g (corresponding to 40.0 mmol) of imidazole were dissolved in 10 mLof dimethylformamide, and cooled to 0° C. Then, 5.50 g (corresponding to20.0 mmol) of t-butyldiphenylchlorosilane was added thereto. After thereaction mixture was stirred at room temperature for 18 hours, asaturated sodium chloride solution was added, and extracted three timeswith ethyl acetate. The combined ethyl acetate layers were dried overanhydrous sodium sulfate, and concentrated under reduced pressure. Theresulting crude product was purified by silica gel column chromatography(elution solvent: hexane/ethyl acetate=10/1) to obtain 7.04 g(corresponding to 19.4 mmol) of 1-bromo-2-(t-butyldiphenylsiloxy)ethane(FIG. 2-8, Step 3).

300 mg (corresponding to 0.893 mmol) of2-(3′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine was dissolved in 5.0mL of dimethylformamide, and 370 mg (corresponding to 2.68 mmol) ofpotassium carbonate was added thereto. Then, 357 mg (corresponding to0.982 mmol) of 1-bromo-2-(t-butyldiphenylsiloxy)ethane was addedthereto. After the reaction mixture was stirred at 90° C. for 2 hours, asaturated sodium chloride solution was added, and extracted three timeswith ethyl acetate. The combined ethyl acetate layers were dried overanhydrous sodium sulfate, and concentrated under reduced pressure. Theresulting crude product was purified by silica gel column chromatography(elution solvent: hexane/ethyl acetate=3/1) to obtain 477 mg(corresponding to 0.771 mmol) of2-[3′-(2″-t-butyldiphenylsiloxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(FIG. 2-8, Step 4).

477 mg (corresponding to 0.771 mmol) of2-[3′-(2″-t-butyldiphenylsiloxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridinewas dissolved in 0.98 mL of tetrahydrofuran, and 0.93 mL of a 1.0 mol/Ltetrahydrofuran solution of tetrabutylammoniumfluoride was addedthereto. After the reaction mixture was stirred at room temperature for15 minutes, ammonium chloride solution was added followed by addition of5.0 mL of water and 2.0 mL of acetonitrile to filter precipitates. Thefiltered precipitates were washed with water and acetonitrile in thisorder to obtain 120 mg (corresponding to 0.316 mmol) of2-[3′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (FIG. 2-8,Step 5).

The NMR measurement results of the resulting2-[3′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (internalstandard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: dimethylsulfoxide-d6; resonance frequency: 500 MHz): δ8.91 (s, 1H), 8.35 (s, 1H), 7.52-7.51 (m, 2H), 7.45 (s, 2H), 7.35 (t,J=8.2 Hz, 1H), 6.93-6.90 (m, 1H), 4.06 (t, J=4.6 Hz, 2H), 3.75 (t, J=4.6Hz, 2H).

Example II-15 Synthesis of6-tributylstannyl-2-[3′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine

70 mg (corresponding to 0.184 mmol) of2-[3′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine obtained inExample II-14 was dissolved in 4.0 mL of dioxane, and 2.0 mL oftriethylamine was added thereto. Then, 0.20 mL (corresponding to 0.39mmol) of bis(tributyltin) and 14.0 mg (a catalytic amount) oftetrakis-triphenylphosphine palladium were added thereto. After thereaction mixture was stirred at 90° C. for 20 hours, the solvent wasdistilled off under reduced pressure. The residue was purified by flashsilica gel column chromatography (elution solvent: hexane/ethylacetate=2/1) to obtain 73.0 mg (corresponding to 0.134 mmol) of6-tributylstannyl-2-[3′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine(FIG. 2-9, Step 1).

The NMR measurement results of the resulting6-tributylstannyl-2-[3′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine(internal standard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl; resonance frequency: 500 MHz): δ 7.99(d, J=0.9 Hz, 1H), 7.82 (s, 1H), 7.64-7.50 (m, 3H), 7.34-7.31 (m, 1H),7.18-7.17 (m, 1H), 6.90-6.87 (m, 1H), 4.20 (t, J=4.3 Hz, 2H), 3.98 (t,J=4.3 Hz, 2H), 1.69-1.48 (m, 6H), 1.39-1.32 (m, 6H), 1.19-1.05 (m, 6H),0.91 (t, J=7.4 Hz, 9H).

Example II-16 Synthesis of2-[3′-(2″-hydroxyethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine

To 60 μL of a mixed solution of6-tributylstannyl-2-[3′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine(concentration: 1 mg/mL) in methanol/dimethylsulfoxide (in a ratio of9/1), 150 μL of 1 mol/L hydrochloric acid, 15 μL of 1 mmol/L sodiumiodide, 250 μL of [¹²³I]sodium iodide of 274 MBq and 15 μL of 10% (w/v)hydrogen peroxide were added. After the mixed solution was left to standat 50° C. for 10 minutes, it was subjected to HPLC under the followingconditions to obtain2-[3′-(2″-hydroxyethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridinefraction.

HPLC conditions:

-   Column: Phenomenex Luna C18 (trade name; manufactured by Phenomenex    Co.; size: 4.6×150 mm)-   Mobile phase: 0.1% trifluoroacetic acid/acetonitrile=20/80 to 0/100    (17 minutes)-   Flow rate: 1.0 mL/min.-   Detector: Ultraviolet visible absorptiometer (Detection wavelength:    282 nm) and radioactivity counter (manufactured by raytest: type    STEFFI)

10 ml of water was added to the fraction. The resulting solution waspassed through a reversed phase column (trade name: Sep-Pak (registeredtrademark) Light C8 Cartridges manufactured by Waters: the packed amountof the packing agent: 145 mg) so that the column adsorbs and collects2-[3′-(2″-hydroxyethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine. Thecolumn was rinsed with 1 mL of water, and then 1 mL of diethyl ether waspassed therethrough to elute2-[3′-(2″-hydroxyethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine. Theamount of radioactivity of the obtained compound was 112.9 MBq at theend of synthesis. Further, the TLC analysis was conducted under thefollowing conditions, and as a result, the radiochemical purity of thecompound was 97%.

TLC analysis conditions:

-   TLC plate: Silica Gel 60 F₂₅₄ (trade name; manufactured by Merck &    Co., Inc.)-   Mobile phase: Chloroform/methanol/triethylamine=100/1/2-   Detector: Rita Star (trade name; manufactured by raytest)

Example II-17 Synthesis of2-[4′-(3″-hydroxypropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(non-radioactive iodinated form)

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 2-10, Step 1).

987 mg (corresponding to 4.55 mmol) of 2-bromo-4′-hydroxyacetophenoneand 1.00 g (corresponding to 4.55 mmol) of 2-amino-5-iodopyridine weredissolved in 50 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 110° C. for 2 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 10 mL of water and 1 mLof methanol. Then, about 10 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 927 mg (corresponding to2.76 mmol) of 2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine (FIG.2-10, Step 2).

Separately, 7.0 g (corresponding to 50.4 mmol) of 2-bromopropanol and6.86 g (corresponding to 101 mmol) of imidazole were dissolved in 50 mLof dimethylformamide, and cooled to 0° C. Then, 7.59 g (corresponding to50.4 mmol) of t-butyldimethylchlorosilane was added thereto. After thereaction mixture was stirred at room temperature for 24 hours, it wassupplemented with a saturated sodium chloride solution, and extractedthree times with diethyl ether. The combined diethyl ether layers weredried over anhydrous magnesium sulfate, and concentrated under reducedpressure. The resulting crude product was purified by vacuumdistillation (100° C., 70 mmHg), to obtain 7.23 g (corresponding to 30.2mmol) of 1-bromo-3-(t-butyldimethylsiloxy)propane (FIG. 2-10, Step 3).

2.00 g (corresponding to 5.95 mmol) of2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine was dissolved in 30.0mL of dimethylformamide, and 2.47 g (corresponding to 17.9 mmol) ofpotassium carbonate was added. Then, 1.51 g (corresponding to 5.95 mmol)of 1-bromo-3-(t-butyldimethylsiloxy)propane was added thereto. After thereaction mixture was stirred at room temperature for 8 days, it wassupplemented with a saturated sodium chloride solution, and extractedthree times with ethyl acetate. The combined ethyl acetate layers weredried over anhydrous sodium sulfate, and concentrated under reducedpressure. The resulting crude product was purified by silica gel columnchromatography (elution solvent: hexane/ethyl acetate=1/1) to obtain1.52 g (corresponding to 2.99 mmol) of2-[4′-(3″-t-butyldimethylsiloxypropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(FIG. 2-10, Step 4).

1.52 g (corresponding to 2.99 mmol) of2-[4′-(3″-t-butyldimethylsiloxypropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridinewas dissolved in 5.0 mL of tetrahydrofuran, and 2.99 mL of a 1.0 mol/Ltetrahydrofuran solution of tetrabutylammoniumfluoride was addedthereto. After the reaction mixture was stirred at room temperature for30 minutes, ammonium chloride solution was added followed by theaddition of 10 mL of water and 5.0 mL of acetonitrile to filterprecipitates. The filtered precipitates were washed with water andacetonitrile in this order, to obtain 1.03 g (corresponding to 2.61mmol) of 2-[4′-(3″-hydroxypropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(FIG. 2-10, Step 5).

The NMR measurement results of the resulting2-[4′-(3″-hydroxypropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine (internalstandard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: dimethylformamide-d6; resonance frequency: 500 MHz): δ8.96 (s, 1H), 8.33 (s, 1H), 7.98 (d, J=8.7 Hz, 2H), 7.46 (s, 2H), 7.06(d, J=8.7 Hz, 2H), 4.63 (t, J=5.0 Hz, 1H), 4.17 (t, J=6.0 Hz, 2H), 3.72(dt, J=5.0, 6.0 Hz, 2H), 1.98 (tt, J=6.0, 6.0 Hz, 2H).

Example II-18 Synthesis of6-tributylstannyl-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine

50 mL of ethyl acetate was added to 28.17 g (corresponding to 126 mmol)of cupric bromide to obtain a suspension, to which a solution of 8.18 g(corresponding to 60.0 mmol) of 4′-hydroxyacetophenone in a mixedsolution of 50 mL of ethyl acetate and 50 mL of chloroform was added.Then, the resulting mixture was refluxed. After 5 hours, the reactionmixture was cooled down to room temperature and filtered. The resultingfiltrate was concentrated under reduced pressure. The residue wasdissolved in ethyl acetate and subjected to decoloring operation withaddition of active charcoal. Then, the resulting solution was filteredand concentrated. The resulting crude product was purified by flashsilica gel column chromatography (elution solvent:chloroform/methanol=20/1), and recrystallized from ethylacetate/petroleum ether, to obtain 7.25 g (corresponding to 33.7 mmol)of 2-bromo-4′-hydroxyacetophenone (FIG. 2-11, Step 1).

2.15 g (corresponding to 10.0 mmol) of 2-bromo-4′-hydroxyacetophenoneand 1.74 g (corresponding to 10.0 mmol) of 2-amino-5-bromopyridine weredissolved in 50 mL of acetonitrile. The resulting solution was refluxedin an oil bath at 105° C. for 6 hours. After the completion of thereaction, the reaction solution was cooled down to room temperature, andprecipitates were filtered and recovered. The precipitates were washedwith acetonitrile and dried under reduced pressure. The resulting crudecrystals were suspended in a mixed solution of 20 mL of water and 20 mLof methanol. Then, about 25 mL of a saturated sodium hydrogencarbonatesolution was added thereto, and the mixture was sonicated for 5 minutesusing an ultrasonic washing machine. Precipitates were filtered andrecovered from the resulting mixture, sufficiently washed with water,and dried under reduced pressure, to obtain 2.41 g (corresponding to8.32 mmol) of 6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine (FIG.2-11, Step 2).

1.45 g (corresponding to 5.0 mmol) of6-bromo-2-(4′-hydroxyphenyl)imidazo[1,2-a]pyridine that was sufficientlydried to remove moisture was dissolved in 50 mL ofN,N-dimethylformamide, and 2.07 g (corresponding to 15.0 mmol) ofpotassium carbonate was added thereto. The mixture was supplemented with680 μL (corresponding to 7.5 mmol) of 3-bromo-1-propanol, and then thesolution was stirred at room temperature for 17 hours. After thecompletion of the reaction, the reaction solution was poured into waterand extracted three times with chloroform. The combined chloroform layerwas washed with a saturated sodium chloride solution, dried overanhydrous sodium sulfate, filtered and concentrated. The resulting crudeproduct was recrystallized from methanol to obtain 1.28 g (correspondingto 3.67 mmol) of6-bromo-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine (FIG.2-11, Step 3).

100 mg (corresponding to 0.288 mmol) of6-bromo-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine wasdissolved in 4.0 mL of dioxane, and 2.0 mL of triethylamine was addedthereto. Then, 0.22 mL (corresponding to 0.43 mmol) of bis(tributyltin)and 22.0 mg (a catalytic amount) of tetrakis-triphenylphosphinepalladium were added thereto. After the reaction mixture was stirred at90° C. for 24 hours, the solvent was distilled off under reducedpressure, and the residue was purified by flash silica gel columnchromatography (elution solvent: hexane/ethyl acetate=3/1) to obtain68.0 mg (corresponding to 0.122 mmol) of6-tributylstannyl-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine(FIG. 2-11, Step 4).

The NMR measurement results of the resulting6-tributylstannyl-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine(internal standard: tetramethylsilane) are shown below.

NMR apparatus employed: JNM-ECP-500 (manufactured by Japan ElectronOptics Laboratory Co., Ltd. (JEOL))

¹H-NMR (solvent: chloroform-dl; resonance frequency: 500 MHz): δ 7.97(s, 1H), 7.88 (d, J=8.3 Hz, 2H), 7.74 (s, 1H), 7.58 (d, J=8.3 Hz, 1H),7.14 (d, J=8.7 Hz, 1H), 6.98 (d, J=8.7 Hz, 2H), 4.18 (t, J=6.0 Hz, 2H)3.89 (t, J=6.0 Hz, 2H), 2.08 (tt, J=6.0, 6.0 Hz, 2H), 1.59-1.49 (m, 6H),1.39-1.31 (m, 6H), 1.18-1.05 (m, 6H), 0.90 (t, J=7.3 Hz, 9H)

Example II-19 Synthesis of2-[4′-(3″-hydroxypropoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine

To 100 μL of a mixed solution of6-tributylstannyl-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine(concentration: 1 mg/mL) in methanol/dimethylsulfoxide (in a ratio of9/1), 80 μL of 2 mol/L hydrochloric acid, 15 μL of 1 mmol/L sodiumiodide, 120 μL of [¹²³i]sodium iodide of 414 MBq and 20 μL of 10% (w/v)hydrogen peroxide were added. After the mixed solution was left to standat 50° C. for 10 minutes, the solution was subjected to HPLC under thefollowing conditions, to obtain2-[3′-(4″-hydroxypropoxy)phenyl]-6-[123]iodoimidazo[1,2-a]pyridinefraction.

HPLC conditions:

-   Column: Phenomenex Luna C18 (trade name; manufactured by Phenomenex    Co.; size: 4.6×150 mm)-   Mobile phase: 0.1% trifluoroacetic acid/acetonitrile=20/80 to 0/100    (17 minutes)-   Flow rate: 1.0 mL/min.-   Detector: Ultraviolet visible absorptiometer (Detection wavelength:    282 nm) and radioactivity counter (manufactured by raytest: type    STEFFI)

10 ml of water was added to the fraction. The resulting solution waspassed through a reversed phase column (trade name: Sep-Pak (registeredtrademark) Light C8 Cartridges manufactured by Waters: the packed amountof the packing agent: 145 mg) so that the column adsorbs and collects2-[4′-(3″-hydroxypropoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine. Thecolumn was rinsed with 1 mL of water, and then 1 mL of diethyl ether waspassed therethrough to elute2-[4′-(3″-hydroxypropoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine. Theamount of radioactivity of the obtained compound was 219 MBq at the endof synthesis. Further, the TLC analysis was conducted under thefollowing conditions, and as a result, the radiochemical purity of thecompound was 97%.

TLC analysis conditions:

-   TLC plate: Silica Gel 60 F₂₅₄ (trade name; manufactured by Merck &    Co., Inc.)-   Mobile phase: Chloroform/methanol/triethylamine=100/1/2-   Detector: Rita Star (trade name; manufactured by raytest)

Example II-20 to II-21, Comparative Example II-7 Measurement ofPartition Coefficient-Based on the Octanol Extraction Method

A diethyl ether solution (Example II-20) of Compound II-4 prepared inExample II-16, a diethyl ether solution (Example II-21) of Compound II-6prepared in Example II-19, and a diethyl ether solution (ComparativeExample II-7) of [¹²³I]-IMPY were each diluted with a 10 mg/mL ascorbicacid-containing physiological saline solution to adjust the radioactiveconcentration to 20-30 MBq/mL. To 2 mL of octanol, 10 μL each of theprepared sample solution was added, and 2 mL of 10 mmol/L phosphatebuffer (pH 7.4) was further added, followed by stirring for 30 seconds.After the mixture was centrifuged with a low-speed centrifuge (2000rpm×60 min.), the octanol layer and the water layer were sampled each inan amount of 1 mL, and subjected to measurement of radioactivity countwith an autowell gamma system (Type: ARC-301B, manufactured by Aloka).Using the obtained radioactivity count, logP_(octanol) was calculated inaccordance with the equation (2-4).

$\begin{matrix}{{\log\; P_{octanol}} = {\log_{10}( \frac{{Radioactivity}\mspace{14mu}{count}\mspace{14mu}{of}\mspace{14mu}{octanol}\mspace{14mu}{layer}}{{Radioactivity}\mspace{14mu}{count}\mspace{14mu}{of}\mspace{14mu}{water}\mspace{14mu}{layer}} )}} & ( {2\text{-}4} )\end{matrix}$

The results are shown in Table 2-6. All compounds showed logP_(octanol)value between 1 and 3. It is known that compounds permeable to BBB showa logP_(octanol) value between 1 and 3 (Douglas D. Dischino et al., J.Nucl. Med., (1983), 24, p. 1030-1038). Thus, it is implied that bothcompounds have a BBB permeability comparable to IMPY.

TABLE 2-6 logP_(octanol) value of the present compound ExperimentCompound logP_(octanol) value Comparative [¹²³I]-IMPY 2.1 Example II-7Example II-20 Compound II-4 2.5 Example II-21 Compound II-6 2.1

Example II-22 to II-23, Comparative Example II-8 Measurement ofTransferability into Brain and Clearance

Using Compound II-4 and Compound II-6, a time course change ofradioactive accumulation in brain of male Wistar rats (7-week old) wasmeasured.

Compound II-4 (Example II-22), Compound II-6 (Example II-23) and asolution of [¹²³I]-IMPY (Comparative Example II-8) prepared above inReference Example II-1 were each diluted with a 10 mg/mL ascorbicacid-containing physiological saline solution to prepare solutions(20-31 MBq/mL in radioactive concentration). 0.05 mL each of theprepared sample solutions was injected under thiopental anesthesia intothe tail vein of the respective Wistar rat (7-week old). The rats weresacrificed by bleeding from abdominal artery, and brains were removedand subjected to measurement of mass of brains and further subjected tomeasurement of radioactivity (hereinafter referred to as A in thisExample) with a single channel analyzer (detector type SP-20manufactured by OHYO KOKEN KOGYO Co., Ltd.) 2, 5, 30 and 60 minutesafter the injection. Further, the radioactivity level of the rest of thewhole body was measured in the same manner as above (hereinafterreferred to as B in this Example). Using these measurement results,radioactive distribution per unit weight of brain (% ID/g) at therespective time points were calculated in accordance with the followingformula (2-5).

Three animals were used for the experiment at the respective timepoints.

$\begin{matrix}{{\%\mspace{14mu}{{ID}/g}} = {\frac{A}{B \times {brain}\mspace{14mu}{weight}} \times 100}} & ( {2\text{-}5} )\end{matrix}$

The results are shown in Table 2-7. As shown in Table 2-7, CompoundsII-4 and II-6 showed a significant accumulation like [¹²³I]-IMPY at thetime point of two minutes after the injection, and then showed atendency to rapidly clear away in 60 minutes. These results suggest thatCompounds II-4 and II-6 possess high transferability to brain and rapidclearance from brain like [¹²³I]-IMPY.

TABLE 2-7 Radioactive distribution in brain of the present compoundafter intravenous injection (rats) Radioactive distribution per unitweight (% ID/g) Compound After 2 min. After 5 min. After 30 min. After60 min. Example Compound 0.56 0.28 0.04 0.01 II-22 II-4 Example Compound0.81 0.56 0.07 0.02 II-23 II-6 Comparative ¹²³I-IMPY 1.19 0.97 0.23 0.09Example II-8

Example II-24 to II-25 Confirmation of Imaging of Amyloid in Brain

(1) Aβ₁₋₄₂ (Wako) was dissolved in phosphate buffer (pH 7.4) and shakenat 37° C. for 72 hours, to obtain 1 mg/mL of a suspension of aggregatedAβ (hereinafter referred to as amyloid suspension in the Examples).

(2) 2.5 μL (corresponding to 25 μg) of the amyloid suspension wasinjected into an amygdaloid nucleus on one side of a male Wistar rat(7-week old). As a control, 2.5 μL of a phosphate buffered physiologicalsaline solution (pH 7.4) was injected into an amygdaloid nucleus on theother side of the rat. The rats were examined 1 day after the injectionof the amyloid suspension and the phosphate buffered physiologicalsaline solution (pH 7.4).

(3) A sample solution (30 MBq/mL in radioactivity concentration, ExampleII-24) in which Compound II-4 was dissolved in a 10 mg/mL ascorbicacid-containing physiological saline solution and a sample solution (30MBq/mL in radioactivity concentration, Example II-25) in which CompoundII-6 was dissolved in a 10 mg/mL ascorbic acid-containing physiologicalsaline solution were prepared. This solution was injected underthiopental anesthesia into the rat through the tail vein (dosage: 0.5mL, dosed radioactivity: 11-15 MBq equivalent).

(4) Brain was removed 60 minutes after the injection to prepare a brainslice of 10 μm in thickness with a microtome (type: CM3050S,manufactured by LEICA). The brain slice was exposed to an imaging platefor 20 hours, and then image analysis was carried out by use of aBio-imaging Analyzer (type: BAS-2500; manufactured by FUJIFILMCorporation).

(5) After the completion of the image analysis using the Bio-imagingAnalyzer, pathological staining with Thioflavin T was carried out toperform imaging by use of a fluorescence microscope (manufactured byNIKON Corporation; type: TE2000-U model; excitation wavelength: 400-440nm; detection wavelength: 470 nm). Thus, it was confirmed that amyloidwas deposited on the slice (FIG. 2-12 and FIG. 2-13).

FIG. 2-12 and FIG. 2-13 show images by autoradiogram and Thioflavin Tstaining of the brain slice of the rat to which amyloid was injectedintracerebrally. As shown in these figures, a marked accumulation ofradioactivity was observed in the amygdaloid nucleus on the side towhich the amyloid suspension was injected, in both cases where CompoundsII-4 and II-6 were administered. On the other hand, no significantaccumulation of radioactivity was observed in the amygdaloid nucleus onthe side to which the physiological saline solution was injected,compared with the other sites. On the autoradiogram, little accumulationof radioactivity was observed at sites other than the site to whichamyloid was injected. From the result of Thioflavin T staining, it wasconfirmed that amyloid was present in the site where radioactivity wasaccumulated (FIG. 2-12 and FIG. 2-13). These results suggest thatCompounds II-4 and II-6 possess a property of accumulating onintracerebral amyloid and a capability of imaging intracerebral amyloid.

Industrial Applicability

The compounds of the present invention can be utilized in the field ofdiagnostics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-1 is a scheme of synthesis of6-tributylstannyl-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine.

FIG. 1-2 is a scheme of synthesis of6-bromo-2-[4′-(3″-p-toluenesulfonyloxypropoxy)phenyl]imidazo[1,2-a]pyridine.

FIG. 1-3 is a scheme of synthesis of6-bromo-2-[4′-(2″-p-toluenesulfonyloxyethoxy)phenyl]imidazo[1,2-a]pyridine.

FIG. 1-4 is a scheme of synthesis of6-bromo-2-[4′-(3″-fluoropropoxy)phenyl]imidazo[1,2-a]pyridine.

FIG. 1-5 is a scheme of synthesis of2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine.

FIG. 1-6 is a scheme of synthesis of6-bromo-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridine.

FIG. 1-7 is a scheme of synthesis of2-[4′-(2″-fluoroethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine.

FIG. 1-8 is a scheme of synthesis of2-[4′-(3″-fluoropropoxy)phenyl]-6-iodoimidazo[1,2-a]pyrimidine.

FIG. 1-9 is a scheme of synthesis of[¹²⁵I]-2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine.

FIG. 1-10 is a scheme of synthesis of2-(4′-hydroxyphenyl)-6-iodoimidazo[1,2-a]pyridine.

FIG. 1-11 is a relationship between amyloid concentration andradioactive concentration in sample solutions.

FIG. 1-12( a) is an autoradiogram of the brain slice after the injectionof Compound I-7, and FIG. 1-12( b) is a fluorescent microscopic image ofthe Thioflavin T stained sample (a magnification of the site to whichthe amyloid suspension was injected).

FIG. 1-13 is a scheme of synthesis of6-tributylstannyl-2-[4′-(2″-fluoroethoxy)phenyl]imidazo[1,2-a]pyridine.

FIG. 1-14( a) is an autoradiogram of the brain slice after the injectionof Compound I-9, and FIG. 1-14( b) is a fluorescent microscopic image ofthe Thioflavin T stained sample (a magnification of the site to whichthe amyloid suspension was injected).

FIG. 2-1 is a scheme of synthesis of2-[4′-(2-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(non-radioactive iodinated form).

FIG. 2-2 is a scheme of synthesis of6-tributylstannyl-2-[4′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine.

FIG. 2-3 is a scheme of synthesis of2-(4′-ethoxyphenyl)-6-iodoimidazo[1,2-a]pyridine (non-radioactiveiodinated form).

FIG. 2-4 is a scheme of synthesis of6-tributylstannyl-2-(4′-ethoxyphenyl)imidazo[1,2-a]pyridine.

FIG. 2-5( a) is an autoradiogram of the brain slice after the injectionof ¹²³I-IMPY, and FIG. 2-5( b) is a fluorescent microscopic image of theThioflavin T stained sample (a magnification of the site to which theamyloid suspension was injected).

FIG. 2-6( a) is an autoradiogram of the brain slice after the injectionof 2-[4′-(2″-hydroxyethoxy)phenyl]-6-[¹²³I]iodoimidazo[1,2-a]pyridine,and FIG. 2-6( b) is a fluorescent microscopic image of the Thioflavin Tstained sample (a magnification of the site to which the amyloidsuspension was injected).

FIG. 2-7( a) is an autoradiogram of the brain slice after the injectionof 2-(4′-ethoxyphenyl)-6-[¹²³I]iodoimidazo[1,2-a]pyridine, and FIG. 2-7(b) is a fluorescent microscopic image of the Thioflavin T stained sample(a magnification of the site to which the amyloid suspension wasinjected).

FIG. 2-8 is a scheme of synthesis of2-[3′-(2″-hydroxyethoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(non-radioactive iodinated form).

FIG. 2-9 is a scheme of synthesis of6-tributylstannyl-2-[3′-(2″-hydroxyethoxy)phenyl]imidazo[1,2-a]pyridine.

FIG. 2-10 is a scheme of synthesis of2-[4′-(3″-hydroxypropoxy)phenyl]-6-iodoimidazo[1,2-a]pyridine(non-radioactive iodinated form).

FIG. 2-11 is a scheme of synthesis of6-tributylstannyl-2-[4′-(3″-hydroxypropoxy)phenyl]imidazo[1,2-a]pyridine.

FIG. 2-12( a) is an autoradiogram of the brain slice after the injectionof Compound II-4, and FIG. 2-12( b) is a fluorescent microscopic imageof the Thioflavin T stained sample (a magnification of the site to whichthe amyloid suspension was injected).

FIG. 2-13( a) is an autoradiogram of the brain slice after the injectionof Compound II-6, and FIG. 2-13( b) is a fluorescent microscopic imageof the Thioflavin T stained sample (a magnification of the site to whichthe amyloid suspension was injected).

1. A compound represented by the following formula (1), or a saltthereof:

wherein A₁, A₂, A3 and A4 each represents a carbon, R¹ is a halogensubstituent, R² is a halogen substituent, and m is an integer of 0 to 2,provided that at least one of R¹ and R² is a radioactive halogensubstituent and R¹ binds to a carbon represented by A₁, A₂, A₃ or A₄. 2.A compound or a salt thereof according to claim 1, wherein R¹ isselected from the group consisting of ¹⁸F, ⁷⁶Br, ¹²³I, ¹²⁴I, ¹²⁵I and¹³¹I.
 3. A compound or a salt thereof according to claim 1, wherein R²is selected from the group consisting of ¹⁸F, ⁷⁶Br, ¹²³I, ¹²⁴I, ¹²⁵I and¹³¹I.
 4. A compound represented by the following formula (2), or a saltthereof:

wherein A₅, A₆, A₇ and A₈ each represents a carbon, R³ is a groupselected from the group consisting of a nitro substituent,trialkylstannyl substituent having alkyl chains with 1 to 4 carbon atomsand a triphenylstannyl group, R⁴ is a group selected from the groupconsisting of a non-radioactive halogen substituent, amethanesulfonyloxy substituent, a trifluoromethanesulfonyloxysubstituent or aromatic sulfonyloxy substituent, and n is an integer of0 to 2, provided that R³ binds to a carbon represented by A₅, A₆, A₇ orA₈.
 5. A compound or a salt thereof according to claim 4, wherein R³ isselected from the group consisting of a nitro substituent, atrimethylstannyl substituent, a tributylstannyl substituent and atriphenylstannyl group.
 6. A diagnostic agent for Alzheimer's disease,which comprises a compound represented by the following formula (1), ora salt thereof:

wherein A₁, A₂, A₃ and A₄ each represents a carbon, R¹ is a halogensubstituent, R² is a halogen substituent, and m is an integer of 0 to 2,provided that at least one of R¹ and R² is a radioactive halogensubstituent and R¹ binds to a carbon represented by A₁, A₂, A₃ or A₄. 7.The low-toxic diagnostic agent for Alzheimer's disease according toclaim 6, wherein R¹ is selected from the group consisting of ¹⁸F, ⁷⁶Br,¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I.
 8. The low-toxic diagnostic agent forAlzheimer's disease according to claim 6, wherein R² is selected fromthe group consisting of ¹⁸F, ⁷⁶Br, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I.